Protection of endogenous therapeutic peptides from peptidase activity through conjugation to blood components

ABSTRACT

A method of synthesizing a modified therapeutic peptide capable of forming a peptidase-stabilized therapeutic peptide conjugate, the peptide having between 3 and 50 amino acids, is k. In a first step of the method, a therapeutic peptide having a carboxy terminal amino acid and amino terminal amino acid is synthesized. In a second step, pairs of cysteine residues present in the therapeutic peptide are sequentially and selectively oxidized to form disulfide bridges in the therapeutic peptide. In a third step, a protecting group is attached to remaining cysteine residues that do not form disulfide bridges in the therapeutic peptide. Finally, the peptide is coupled to a reactive group capable of reacting with amino groups, hydroxyl groups or thiol groups on a blood component to form a covalent bond therewith.

This application claims benefit of Provision Appls. 60/134,406 filed May17, 1999, Provisional No. 60/153,406 filed Sep. 10, 1999 and ProvisionalNo. 60/159,783 filed Oct. 15, 1999.

FIELD OF THE INVENTION

This invention relates to modified therapeutic peptides. In particular,this invention relates to protection of endogenous therapeutic peptidesfrom peptidase activity through a modification that enables the peptideto selectively conjugate to blood components, thus protecting thepeptide from peptidase activity and increasing the duration of action ofthe therapeutic peptide for the treatment of various disorders.

BACKGROUND OF THE INVENTION

Many endogenous peptides have been described as key components ofbiological processes. Some of these peptides have been identified as keytherapeutic agents for the management of various disorders. In general,endogenous peptides are more desirable as therapeutic agents thansynthetic peptides with non-native sequences, because they do notproduce an immune response due to their endogenous character. Inaddition, endogenous peptides are highly specific for their targetreceptors and are easy to synthesize and manufacture. However, a majordifficulty with the delivery of such therapeutic peptides is their shortplasma half-life, mainly due to rapid serum clearance and proteolyticdegradation via the action of peptidases.

Peptidases break a peptide bond in peptides by inserting a watermolecule across the bond. Generally, most peptides are broken down bypeptidases in the body in a manner of a few minutes or less. Inaddition, some peptidases are specific for certain types of peptides,making their degradation even more rapid. Thus, if a peptide is used asa therapeutic agent, its activity is generally reduced as the peptidequickly degrades in the body due to the action of peptidases.

One way to overcome this disadvantage is to administer large dosages ofthe therapeutic peptide of interest to the patient so that even if someof the peptide is degraded, enough remains to be therapeuticallyeffective. However, this method is quite uncomfortable for the patient.Since most therapeutic peptides cannot be administered orally, thetherapeutic peptide would have to be either constantly infused,frequently administered by intravenous injections, or administeredfrequently by the inconvenient route of subcutaneous injections. Theneed for frequent administration also results in many potential peptidetherapeutics having an unacceptably high projected cost per treatmentcourse. The presence of large amounts of degraded peptide may alsogenerate undesired side effects.

Discomfort in administration and high costs are two reasons why mosttherapeutic peptides with attractive bioactivity profiles are notdeveloped as drug candidates. Instead, these therapeutic peptides areused as templates for the development of peptidomimetic compounds tosubstitute for the therapeutic peptide. Biotechnology and largepharmaceutical firms frequently undertake lengthy and expensiveoptimization programs to attempt to develop non-peptide, organiccompounds which mimic the activity seen with therapeutic peptideswithout incurring an unacceptable side effect profile. For example,cyclic peptides, peptidomimetics and small molecules coming fromexpensive SAR (Structure Activity Relationship) and molecular modelingstudies have led to the development of an incredible amount of peptidemimics. However, these peptide mimics in no way reflect the exactoriginal biological nature of the therapeutic peptide, and thus areinferior to the endogenous therapeutic peptide as therapeutic agents.

An alternative to creating peptide mimics is to block the action ofpeptidases to prevent degradation of the therapeutic peptide or tomodify the therapeutic peptides in such a way that their degradation isslowed down while still maintaining biological activity. Such methodsinclude conjugation with polymeric materials such as dextrans, polyvinylpyrrolidones, glycopeptides, polyethylene glycol and polyamino acids,conjugation with adroitin sulfates, as well as conjugation withpolysaccharides, low molecular weight compounds such as aminolethicin,fatty acids, vitamin B₁₂, and glycosides. These conjugates, however, arestill often susceptible to protease activity. In addition, thetherapeutic activity of these peptides is often reduced by the additionof the polymeric material. Finally, there is the risk of the conjugatesgenerating an immune response when the material is injected in vivo.Several methods include ex vivo conjugation with carrier proteins,resulting in the production of randomized conjugates. Since conjugatesare difficult to manufacture, and their interest is limited bycommercial availability of the carriers, as well as by their poorpharmaco economics.

There is thus a need for novel methods to modify therapeutic peptides toprotect them from peptidase activity and to provide longer duration ofaction in vivo, while maintaining low toxicity yet retaining thetherapeutic advantages of the modified peptides.

SUMMARY OF THE INVENTION

This invention is directed to overcoming the problem of peptidedegradation in the body by modifying the therapeutic peptide of interestand attaching it to protein carriers, such that the action of peptidasesis prevented, or slowed down. More specifically, this invention relatesto novel chemically reactive derivatives of therapeutic peptides thatcan react with available functionalities on blood proteins to formcovalent linkages, specifically a therapeutic peptide-maleimidederivative. The invention also relates to novel chemically reactivederivatives or analogs of such therapeutic peptides. The inventionadditionally pertains to the therapeutic uses of such compounds.

The present invention is directed to modifying and attaching therapeuticpeptides to protein carriers, preferentially albumin, through in vivo orex vivo technology to prevent or reduce the action of peptidases byvirtue of a synthetic modification on the first residue to be cleaved.Therapeutic peptides are usually active at the N-terminus portion, atthe C-terminus portion, or in an interior portion of the peptide chain.Using the technology of this invention, a site other than the activeportion of a therapeutic peptide is modified with certain reactivegroups. These reactive groups are capable of forming covalent bonds withfunctionalities present on blood components. The reactive group isplaced at a site such that when the therapeutic peptide is bonded to theblood component, the peptide retains a substantial proportion of theparent compound's activity.

The modification of the therapeutic peptide through the chemicalmodification used in the invention is done in such a way that all ormost of the peptide specificity is conserved despite attachment to ablood component. This therapeutic peptide-blood component complex is nowcapable of traveling to various body regions without and being degradedby peptidases, with the peptide still retaining its therapeuticactivity. The invention is applicable to all known therapeutic peptidesand is easily tested under physiological conditions by the directcomparison of the pharmacokinetic parameters for the free and themodified therapeutic peptide.

The present invention is directed to a modified therapeutic peptidecapable of forming a peptidase stablilized therapeutic peptide composedof between 3 and 50 amino acids. The peptide has a carboxy terminalamino acid, an amino terminal amino acid, a therapeutically activeregion of amino acids and a less therapeutically active region of aminoacids. The peptide comprises a reactive group which reacts with aminogroups, hydroxyl groups, or thiol groups on blood components to form astable covalent bond and thereby forms the peptidase stabilizedtherapeutic peptide. In the peptide of the invention the reactive groupis selected from the group consisting of succinimidyl and maleimidogroups and the reactive group is attached to an amino acid positioned inthe less therapeutically active region of amino acids.

In one embodiment, the therapeutically active region of the peptideincludes the carboxy terminal amino acid and the reactive group isattached to said amino terminal amino acid.

In another embodiment, the therapeutically active region of the peptideincludes the amino terminal amino acid and the reactive group isattached to the carboxy terminal amino acid.

In another embodiment, the therapeutically active region of the peptideincludes the carboxy terminal amino acid and the reactive group isattached to an amino acid positioned between the amino terminal aminoacid and the carboxy terminal amino acid.

In yet another embodiment, the therapeutically active region includesthe amino terminal amino acid and the reactive group is attached to anamino acid positioned between the amino terminal amino acid and thecarboxy terminal amino acid.

The present invention is also directed to a method of synthesizing themodified therapeutic peptide. The method comprises the following steps.In the first step, if the therapeutic peptide does not contain acysteine, then the peptide is synthesized from the carboxy terminalamino acid and the reactive group is added to the carboxy terminal aminoacid. Alternatively, a terminal lysine is added to the carboxy terminalamino acid and the reactive group is added to the terminal lysine. Inthe second step, if the therapeutic peptide contains only one cysteine,then the cysteine is reacted with a protective group prior to additionof the reactive group to an amino acid in the less therapeuticallyactive region of the peptide. In the third step, if the therapeuticpeptide containe two cysteines as a disulfide bridge, then the twocysteines are oxidized and the reactive group is added to the aminoterminal amino acid, or to the carboxy terminal amino acid, or to anamino acid positioned between the carboxy terminal amino acid and theamino terminal amino acid of the therapeutic peptide. In the fourthstep, if the therapeutic peptide contains more than two cysteines asdisulfide bridges, the cysteines are sequentially oxidized in thedisulfide bridges and the peptide is purified prior to the addition ofthe reactive groups to the carboxy terminal amino acid.

The present invention is also directed to a method for protecting atherapeutic peptide from peptidase activity in vivo, the peptide beingcomposed of between 3 and 50 amino acids and having a carboxy terminusand an amino terminus and a carboxy terminal amino acid amino acid andan amino terminal amino acid. The method comprises the following steps:

(a) modifying the peptide by attaching a reactive group to the carboxyterminal amino acid, to the amino terminal amino acid, or to an aminoacid located between the amino terminal amino acid and the carboxyterminal amino acid, such that the modified peptide is capable offorming a covalent bond in vivo with a reactive functionality on a bloodcomponent;

(b) forming a covalent bond between the reactive group and a reactivefunctionality on a blood component to form a peptide-blood componentconjugate, thereby protecting the peptide from peptidase activity; and

(c) analyzing the stability of the peptide-blood component conjugate toassess the protection of the peptide from peptidase activity. Thesesteps may be performed either in vivo or ex vivo.

The present invention is also directed to a method for protecting atherapeutic peptide from peptidase activity in vivo, the peptide beingcomposed of between 3 and 50 amino acids and having a therapeuticallyactive region of amino acids and a less therapeutically active region ofamino acids. The method comprises the following steps:

(a) determining the therapeutically active region of amino acids;

(b) modifying the peptide at an amino acid included in the lesstherapeutically active region of amino acids by attaching a reactivegroup to the amino acid to form a modified peptide, such that themodified peptide has therapeutic activity and is capable of forming acovalent bond in vivo with a reactive functionality on a bloodcomponent;

(c) forming a covalent bond between the reactive entity and a reactivefunctionality on a blood component to form a peptide-blood componentconjugate, thereby protecting the peptide from peptidase activity; and

(d) analyzing the stability of the peptide-blood component conjugate toassess the protection of the peptide from peptidase activity. Thesesteps may be performed either in vivo or ex vivo.

The peptides useful in the compositions and methods of the presentinvention include, but are not limited to, the peptides presented in SEQID NO:1 to SEQ ID NO:1617.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

To ensure a complete understanding of the invention, the followingdefinitions are provided:

Reactive Groups:

Reactive groups are entities capable of forming a covalent bond. Suchreactive groups are coupled or bonded to a therapeutic peptide ofinterest. Reactive groups will generally be stable in an aqueousenvironment and will usually be carboxy, phosphoryl, or convenient acylgroup, either as an ester or a mixed anhydride, or an imidate, therebycapable of forming a covalent bond with functionalities such as an aminogroup, a hydroxy or a thiol at the target site on mobile bloodcomponents. For the most part, the esters will involve phenoliccompounds, or be thiol esters, alkyl esters, phosphate esters, or thelike. Reactive groups include succimidyl and maleimido groups.

Functionalities:

Functionalities are groups on blood components, including mobile andfixed proteins, to which reactive groups on modified therapeuticpeptides react to form covalent bonds. Functionalities usually includehydroxyl groups for bonding to ester reactive groups, thiol groups forbonding to maleimides, imidates and thioester groups; amino groups forbonding to activated carboxyl, phosphoryl or any other acyl groups onreactive groups.

Blood Components:

Blood components may be either fixed or mobile. Fixed blood componentsare non-mobile blood components and include tissues, membrane receptors,interstitial proteins, fibrin proteins, collagens, platelets,endothelial cells, epithelial cells and their associated membrane andmembraneous receptors, somatic body cells, skeletal and smooth musclecells, neuronal components, osteocytes and osteoclasts and all bodytissues especially those associated with the circulatory and lymphaticsystems. Mobile blood components are blood components that do not have afixed situs for any extended period of time, generally not exceeding 5,more usually one minute. These blood components are notmembrane-associated and are present in the blood for extended periods oftime and are present in a minimum concentration of at least 0.1 μg/ml.Mobile blood components include serum albumin, transferrin, ferritin andimmunoglobulins such as IgM and IgG. The half-life of mobile bloodcomponents is at least about 12 hours.

Protective Groups:

Protective groups are chemical moieties utilized to protect peptidederivatives from reacting with themselves. Various protective groups aredisclosed herein and in U.S. Pat. No. 5,493,007 which is herebyincorporated by reference. Such protective groups include acetyl,fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc),benzyloxycarbonyl (Cbz), and the like. The specific protected aminoacids are depicted in Table 1.

Linking Groups:

Linking groups are chemical moieties that link or connect reactivegroups to therapeutic peptides. Linking groups may comprise one or morealkyl groups, alkoxy group, alkenyl group, alkynyl group or amino groupsubstituted by alkyl groups, cycloalkyl group, polycyclic group, arylgroups, polyaryl groups, substituted aryl groups, heterocyclic groups,and substituted heterocyclic groups. Linking groups may also comprisepoly ethoxy aminoacids such as AEA ((2-amino)ethoxy acetic acid) or apreferred linking group AEEA ([2-(2-amino)ethoxy)]ethoxy acetic acid). Apreferred linking group is aminoethoxyethoxyacetic acid (AEEA).

Sensitive Functional Groups

A sensitive functional group is a group of atoms that represents apotential reaction site on a therapeutic peptide. If present, asensitive functional group may be chosen as the attachment point for thelinker-reactive group modification. Sensitive functional groups includebut are not limited to carboxyl, amino, thiol, and hydroxyl groups.

Modified Therapeutic Peptides

A modified therapeutic peptide peptide is a therapeutic peptide that hasbeen modified by attaching a reactive group, and is capable of forming apeptidase stabalized peptide through conjugation to blood components.The reactive group may be attached to the therapeutic peptide either viaa linking group, or optionally without using a linking group. It is alsocontemplated that one or more additional amino acids may be added to thetherapeutic peptide to facilitage the attachment of the reactive group.Modified peptides may be administered in vivo such that conjugation withblood components occurs in vivo, or they may be first conjugated toblood components in vitro and the resulting peptidase stabalized peptide(as defined below) administered in vivo. The terms “modified therapeuticpeptide” and “modified peptide” may be used interchangeably in thisapplication.

Peptidase Stabalized Therapeutic Peptides

A peptidase stabalized therapeutic peptide is a modified peptide thathas been conjugated to a blood component via a covalent bond formedbetween the reactive group of the modified peptide and thefunctionalities of the blood component, with or without a linking group.Peptidase stabalized peptides are more stable in the presence ofpeptidases in vivo than a non-stabalized peptide. A peptidase stabalizedtherapeutic peptide generally has an increased half life of at least10-50% as compared to a non-stabalized peptide of identical sequence.Peptidase stability is determined by comparing the half life of theunmodified therapeutic peptide in serum or blood to the half life of amodified counterpart therapeutic peptide in serum or blood. Half life isdetermined by sampling the serum or blood after administration of themodified and non-modified peptides and determining the activity of thepeptide. In addition to determining the activity, the length of thetherapeutic peptide may also be measured.

Therapeutic Peptides

As used in this invention, therapeutic peptides are amino acid chains ofbetween 2-50 amino acids with therapeutic activity, as defined below.Each therapeutic peptide has an amino terminus (also referred to asN-terminus or amino terminal amino acid), a carboxyl terminus (alsoreferred to as C-terminus terminal carboxyl terminal amino acid) andinternal amino acids located between the amino terminus and the carboxylterminus. The amino terminus is defined by the only amino acid in thetherapeutic peptide chain with a free α-amino group. The carboxylterminus is defined by the only amino acid in the therapeutic peptidechain with a free α-carboxyl group.

Therapeutic peptides used in the present invention contain atherapeutically active region generally located at the amino terminus,at the carboxyl terminus, or at an internal amino acid. Thetherapeutically active region may be identified using blind or structureactivity relationship (SAR) driven substitution, as defined in moredetail in this application. SAR is an analysis which defines therelationship between the structure of a molecule and its pharmacologicalactivity for a series of compounds. Alternatively, where thetherapeutically active region has previously been defined and isavailable in the literature, it may be obtained by referring toreferences such as scientific journals. Knowledge of the location of thetherapeutically active region of the peptide is important for modifyingthe therapeutic peptide, as defined in more detail below.

Therapeutic peptides used in this invention also contain a lesstherapeutically active region generally located at the amino terminus,at or near the carboxyl terminus, or at or near an internal amino acid.The less therapeutically active region is a region of amino acids thatdoes not coincide with the therapeutically active region of thetherapeutic peptide. The less therapeutically active region is generallylocated away from the therapeutically active region, such thatmodification at the less therapeutically active region does notsubstantially affect the therapeutic activity of the therapeuticpeptide. For example, if the therapeutically active region is located atthe amino terminus, the therapeutic peptide will be modified at eitherthe carboxyl terminus or at an internal amino acid. Alternatively, ifthe therapeutically active region is located at the carboxyl terminus,the therapeutic peptide will be modified at either the amino terminus orat an internal amino acid. Finally, if the therapeutically active regionis located at an internal region, the therapeutic peptide will bemodified at either the amino terminus or the carboxyl terminus.

“Therapeutic activity” is any activity directed toward healing or curinga biological disorder in a patient. Examples of said therapeuticpeptides include pituitary hormones such as vasopressin, oxytocin,melanocyte stimulating hormones, adrenocorticotropic hormones, growthhormones; hypothalamic hormones such as growth hormone releasing factor,corticotropin releasing factor, prolactin releasing peptides,gonadotropin releasing hormone and its associated peptides, luteinizinghormone release hormones, thyrotropin releasing hormone, orexin, andsomatostatin; thyroid hormones such as calcitonins, calcitoninprecursors, and calcitonin gene related peptides; parathyroid hormonesand their related proteins; pancreatic hormones such as insulin andinsulin-like peptides, glucagon, somatostatin, pancreatic polypeptides,amylin, peptide YY, and neuropeptide Y; digestive hormones such asgastrin, gastrin releasing peptides, gastrin inhibitory peptides,cholecystokinin, secretin, motilin, and vasoactive intestinal peptide;natriuretic peptides such as atrial natriuretic peptides, brainnatriuretic peptides, and C-type natriuretic peptides; neurokinins suchas neurokinin A, neurokinin B, and substance P; renin related peptidessuch as renin substrates and inhibitors and angiotensins; endothelins,including big endothelin, endothelin A receptor antagonists, andsarafotoxin peptides; and other peptides such as adrenomedullinpeptides, allatostatin peptides, amyloid beta protein fragments,antibiotic and antimicrobial peptides, apoptosis related peptides, bagcell peptides, bombesin, bone Gla protein peptides, CART peptides,chemotactic peptides, cortistatin peptides, fibronectin fragments andfibrin related peptides, FMRF and analog peptides, galanin and relatedpeptides, growth factors and related peptides, Gtherapeuticpeptide-binding protein fragments, guanylin and uroguanylin, inhibinpeptides, interleukin and interleukin receptor proteins, lamininfragments, leptin fragment peptides, leucokinins, mast celldegranulating peptides, pituitary adenylate cyclase activatingpolypeptides, pancreastatin, peptide T, polypeptides, virus relatedpeptides, signal transduction reagents, toxins, and miscellaneouspeptides such as adjuvant peptide analogs, alpha mating factor,antiarrhythmic peptide, antifreeze polypeptide, anorexigenic peptide,bovine pineal antireproductive peptide, bursin, C3 peptide P16, tumornecrosis factor, cadherin peptide, chromogranin A fragment,contraceptive tetrapeptide, conantokin G, conantokin T, crustaceancardioactive peptide, C-telopeptide, cytochrome b588 peptide, decorsin,delicioius peptide, delta-sleep-inducing peptide, diazempam-bindinginhibitor fragment, nitric oxide synthase blocking peptide, OVA peptide,platelet calpain inhibitor (P1), plasminogen activator inhibitor 1,rigin, schizophrenia related peptide, serum thymic factor, sodiumpotassium Atherapeutic peptidease inhibiro-1, speract, sperm activatingpeptide, systemin, thrombin receptor agonist, thymic humoral gamma2factor, thymopentin, thymosin alpha 1, thymus factor, tuftsin,adipokinetic hormone, uremic pentapeptide and other therapeuticpeptides.

Taking into account these definitions, the focus of this invention is tomodify therapeutic peptides to protect them from peptidase activity invivo and thereby extend the effective therapeutic life of thetherapeutic peptide in question as compared to administration of thepeptide per se to a patient.

1. Therapeutic Peptides Used in the Present Invention

Peptide fragments chosen from the determined amino acid sequence of atherapeutic peptide as provided in the attached SEQUENCE LISTINGconstitute the starting point in the development comprising the presentinvention. The peptides range from 2 to 50 amino acids in length. Theinterchangeable terms “peptide fragment” and “peptide moiety” are meantto include both synthetic and naturally occurring amino acid sequencesderivable from a naturally occurring amino acid sequence.

In one embodiment, peptide and peptide fragments are synthesized byconventional means, either by bench-top methods or by automated peptidesynthesis machines as discussed in detail below. However, it is alsopossible to obtain fragments of the peptides by fragmenting thenaturally occurring amino acid sequence, using, for example, aproteolytic enzyme. Further, it is possible to obtain the desiredfragments of the therapeutic peptide through the use of recombinant DNAtechnology, as disclosed by Maniatis, T., et al., Molecular Biology: ALaboratory Manual, Cold Spring Harbor, N.Y. (1982), which is herebyincorporated by reference. The use of other new modifications toexisting methodologies is also contemplated.

The present invention includes peptides which are derivable from thenaturally occuring sequence of the therapeutic peptide. A peptide issaid to be “derivable from a naturally occurring amino acid sequence” ifit can be obtained by fragmenting a naturally occurring sequence, or ifit can be synthesized based upon a knowledge of the sequence of thenaturally occurring amino acid sequence or of the genetic material (DNAor RNA) which encodes this sequence. Included within the scope of thepresent invention are those molecules which are said to be “derivatives”of a peptide. Such a “derivative” has the following characteristics: (1)it shares substantial homology with the therapeutic peptide or asimilarly sized fragment of the peptide and (2) it is capable offunctioning with the same therapeutic activity as the peptide.

A derivative of a peptide is said to share “substantial homology” withthe peptide if the amino acid sequences of the derivative is at least80%, and more preferably at least 90%, and most preferably at least 95%,the same as that of either the peptide or a fragment of the peptidehaving the same number of amino acid residues as the derivative.

The derivatives of the present invention include fragments which, inaddition to containing a sequence that is substantially homologous tothat of a naturally occurring therapeutic peptide may contain one ormore additional amino acids at their amino and/or their carboxy terminias discussed in detail below. Thus, the invention pertains topolypeptide fragments of the therapeutic peptide that may contain one ormore amino acids that may not be present in a naturally occurringtherapeutic peptide sequence provided that such fragments have atherapeutic activity which exceeds that of the therapeutic peptide.

Similarly, the invention includes polypeptide fragments which, althoughcontaining a sequence that is substantially homologous to that of anaturally occurring therapeutic peptide, may lack one or more additionalamino acids at their amino and/or their carboxy termini that arenaturally found on the therapeutic peptide. Thus, the invention pertainsto polypeptide fragments of the therapeutic peptide that may lack one ormore amino acids that are normally present in the naturally occurringpeptide sequence provided that such polypeptides have a therapeuticactivity which exceeds that of the therapeutic peptide.

The invention also encompasses the obvious or trivial variants of theabove-described fragments which have inconsequential amino acidsubstitutions (and thus have amino acid sequences which differ from thatof the natural sequence) provided that such variants have an activitywhich is substantially identical to that of the above-describedderivatives. Examples of obvious or trivial substitutions include thesubstitution of one basic residue for another (i.e. Arg for Lys), thesubstitution of one hydrophobic residue for another (i.e. Leu for Ile),or the substitution of one aromatic residue for another (i.e. Phe forTyr), etc.

As is known in the art, the amino acid residues may be in theirprotected or unprotected form, using appropriate amino or carboxylprotecting groups as discussed in detail below. The variable lengthpeptides may be in the form of the free amines (on the N-terminus), oracid-addition salts thereof. Common acid addition salts are hydrohalicacid salts, i.e., HBr, HI, or, more preferably, HCl. Useful cations arealkali or alkaline earth metallic cations (i.e., Na, K, Li, Ca, Ba,etc.) or amine cations (i.e., tetraalkylammonium, trialkylammonium,where alkyl can be C₁C₁₂).

Any peptide having a therapeutic activity may be used in this invention.The following list of peptides provides examples of peptides that may beused in this invention, but is not exhaustive and in no way limits thenumber or type of peptides that may be used in this invention. Thesetherapeutic peptides and fragments produced from these peptides may bemodified according to the present invention, and used therapeutically inthe body.

A. Pituitary Hormones (SEQ ID NOS: 1-72)

Adrenocortiocotropic Hormones (ACTH, aka Corticotropin) (SEQ ID NOS:1-22)

The endocrine functions of the adrenal cortex are regulated by ananterior pituitary hormone, ACTH. ACTH, a 39-amino acid peptide isgenerated in the corticotrophic cells of the anterior pituitary underthe control of corticotropin releasing factor. ACTH is derived bypost-translational modification from a 241-amino acid precursor known aspro-opiomelanocortin (POMC).

The biological role of ACTH is to maintain the bulk and the viability ofthe adrenal cortex and to stimulate the production of adrenal cortexsteroids, principally cortisol and costicosterone. The mechanism ofaction of ACTH involves binding to the ACTH receptor followed byactivation of adenylate cyclase, elevation of cyclic AMP (cAMP), andincreased protein kinase A (PKA) activity of adrenal cortex tissue. Themain effect of these events is to increase the activity of a sidechain-cleaving enzyme, which converts cholesterol to pregnenolone.Because of the distribution of enzymes in the various adrenal cortexsubdivisions, the principal physiological effect of ACTH is productionof the glucocorticosteroids.

Aside from its function controlling adrenal cortical activity, ACTHappears to have diverse biological roles including modulation ofendocrine and exocrine glands, temperature regulation and influences onnerve regeneration and development. In addition, ACTH and its fragmentsaffect motivation, learning, and behavior. The use of ACTH as atherapeutic agent may thus help the control of these functions. ACTHrelease from the anterior pituitary is mediated by corticotropinreleasing factor (CRF).

Growth Hormone Peptides (SEQ ID NOS: 23-24, 45)

Human placental lactogen (hPL), growth hormones, and prolactin (Prl)comprise the growth hormone family. All have about 200 amino acids, 2disulfide bonds, and no glycosylation. Although each has specialreceptors and unique characteristics to their activity, they all possessgrowth-promoting and lactogenic activity. Mature GH (22,000 daltons) issynthesized in acidophilic pituitary somatotropes as a singlepolypeptide chain. Because of alternate RNA splicing, a small amount ofa somewhat smaller molecular form is also secreted.

There are a number of genetic deficiencies associated with GH.GH-deficient dwarfs lack the ability to synthesize or secrete GH, andthese short-statured individuals respond well to GH therapy. Pygmieslack the IGF-1 response to GH but not its metabolic effects; thus inpygmies the deficiency is post-receptor in nature. Finally, Laron dwarfshave normal or excess plasma GH, but lack liver GH receptors and havelow levels of circulating IGF-1. The defect in these individuals isclearly related to an inability to respond to GH by the production ofIGF-1. The production of excessive amounts of GH before epiphysealclosure of the long bones leads to gigantism, and when GH becomesexcessive after epiphyseal closure, acral bone growth leads to thecharacteristic features of acromegaly. Using GH as a therapeutic agentwould aid in treating these disorders, and potentially stimulate growthin other cases of short stature with low or normal GH levels.

Melanocyte Stimulating Hormones (MSH) (SEQ ID NOS: 25-39)

Melanocyte stimulating hormone (MSH) is generated in the intermediarypituitary under the control of dopamine. MSH may have importantphysiological roles in the control of vertebrate pigment cellmelanogenesis, neural functioning related to learning and behavior, andfetal development. See Sawyer, T. K. et al., Proc. Nat. Acad. Sci USA,79, 1751 (1982).

Oxytocin (SEQ ID NOS: 40-44)

Oxytocin is involved in the enhancement of lactation, contraction of theuterus, and relaxation of the pelvis prior to childbirth. Oxytocinsecretion in nursing women is stimulated by direct neural feedbackobtained by stimulation of the nipple during suckling. Its physiologicaleffects include the contraction of mammary gland myoepithelial cells,which induces the ejection of milk from mammary glands, and thestimulation of uterine smooth muscle contraction leading to childbirth.Oxytocin causes myoepithelial cells surrounding secretory acini ofmammary glands to contract, pushing milk through ducts. In addition, itstimulates the release of prolactin, and prolactin is trophic on thebreast and stimulates acinar formation of milk. A conjugated oxytocincould thus be used to aid lactation and help relax the pelvis prior tobirth. It could also be used to prevent post partum uterine hemorrage.

Vasopressin (ADH) (SEQ ID NOS: 46-72)

Vasopressin is also known as antidiuretic hormone (ADH), because it isthe main regulator of body fluid osmolarity, causing antidiuresis andincrease in blood pressure. Vasopressin binds plasma membrane receptorsand acts through G-proteins to activate the cyclic AMP/protein kinase A(cAMP/PKA) regulatory system. The secretion of vasopressin is regulatedin the hypothalamus by osmoreceptors, which sense water concentrationand stimulate increased vasopressin secretion when plasma osmolarityincreases. The secreted vasopressin increases the reabsorption rate ofwater in kidney tubule cells, causing the excretion of urine that isconcentrated in Na⁺ and thus yielding a net drop in osmolarity of bodyfluids. Vasopressin deficiency leads to watery urine and polydipsia, acondition known as diabetes insipidus. Using conjugated vasopressin orvasopressin fragments would thus prevent these disorders and allow theregular maintenance of the body's osmolarity.

B. Hypothalamic Hormones (Releasing Factors)

Corticotropin Releasing Factor (CRF) & Related Peptides (SEQ ID NOS:73-102)

Corticotrophin-releasing factor (CRF), a 41 amino acid peptide, plays asignificant role in coordinating the overall response to stress throughactions both in the brain and the periphery. In the brain, CRF isproduced and secreted primarily from parvocellular neurons of theparaventricular hypothalamic nucleus. From there, the CRF-containingneurons project to the portal capillary zone of the median eminence andact to stimulate the secretion of adrenocorticotrophic hormone (ACTH),beta-endorphin, and other proopiomelanocortin (POMC)-derived peptidesfrom the pituitary gland. The subsequent ACTH-induced release of adrenalglucocorticoids represents the final stage in thehypothalamic-pituitary-adrenal axis (HPA), which mediates the endocrineresponse to stress. Besides its neuroendocrine role, CRF also functionsas a neurotransmitter and neuromodulator to elicit a wide spectrum ofautonomic, behavioral and immune effects to physiological,pharmacological, and pathological stimuli.

Clinical studies indicated that CRF hypersecretion is associated withvarious diseases, such as major depression, anxiety-related illness,eating disorder, as well as inflammatory disorder. Low levels of CRFhave been found in Alzheimer's disease, dementias, obesity, and manyendocrine diseases. Therefore, the use of CRF as a therapeutic agent tocounter the effects associated with high levels or low levels of CRFwill provide a basis for the treatment of diseases that are associatedwith abnormal CRF levels. Several peptide antagonists and nonpeptideantagonists have been discovered and widely studied, including a-helicalCRF(9-41), Astressin, D-PheCRF(12-41) (peptide antagonist) and CP-154526(nonpeptide antagonist). These CRF antagonists may provide a novel agentfor treatment of depression, anxiety and other CRF related illnesses.Conjugated CRF peptides could thus be used to maintain adrenal healthand viability during long term steroid use or as anti-inflamatoryagents.

Gonadotropin Releasing Hormone Associated Peptides (GAP) (SEQ ID NOS:103-110)

GAP is contained in the precursor molecule to gonadotropin-releasinghormone (GnRH). GAP has prolactin inhibiting properties. Gn-RH is ahormone secreted by the hypothalamus that stimulate the release ofgonadotrophic hormones follicle stimulating hormone (FSH) andluteinizing hormone (LH). Low levels of circulating sex hormone reducefeedback inhibition on GnRH synthesis, leading to elevated levels of FSHand LH. The latter peptide hormones bind to gonadal tissue, resulting insex hormone production via cyclic AMP (cAMP) and protein kinase A (PKA)mediated pathways. A conjugated GnRM could be used to aid fertility, oras a contraceptive in either males or females. This agent would have usein animals as well as humans.

Growth Hormone Releasing Factor (GRF) (SEQ ID NOS: 111-134)

GRF is a hypothalamic peptide that plays a critical role in controllingthe synthesis and secretion of growth hormone in the anterior pituitary.Some structurally unrelated short peptides have also been reported toelicit growth hormone secretion by a different mechanism.

Under the influence of GRF, growth hormone is released into the systemiccirculation, causing the target tissue to secrete IGF-1. Growth hormonealso has other more direct metabolic effects; it is both hyperglycemicand lipolytic. The principal source of systemic IGF-1 is the liver,although most other tissues secrete and contribute to systemic IGF-1.Liver IGF-1 is considered to be the principal regulator of tissuegrowth. In particular, the IGF-1 secreted by the liver is believed tosynchronize growth throughout the body, resulting in a homeostaticbalance of tissue size and mass. IGF-1 secreted by peripheral tissues isgenerally considered to be autocrine or paracrine in its biologicalaction. The use of a conjugated GRF as a therapeutic agent to increaseGH release, would then help treat disorders involving growth functionsregulated by GRF.

Lutenizing Hormone Release Hormones (LH-RH) (SEQ ID NOS: 135-161)

Luteinizing hormone releasing hormone is the key mediator in theneuroregulation of the secretion of gonadotropins, luteinizing hormone(LH) and follicle stimulating hormone (FSH). LH-RH can modify sexualbehavior by regulating plasma gonadotropin and sex steroid levels. SeeVale, W. W. et al., Peptides, Structure and Biological Function,Proceedings of the Sixth American Peptide Symposium, Gross, E. andMeienhofer, M., eds., 781 (1979). A conjugated LH-RH agent could be usedto stimulate ovulation in humans or animals as an aid to fertility.

Orexins (SEQ ID NOS: 162-164)

Orexins are a family of neuropeptides from the hypothalamus that havebeen recently discovered and characterized. Orexins stimulate appetiteand food consumption. Their genes are expressed bilaterally andsymmetrically in the lateral hypothalamus, which was earlier determinedto be the “feeding center” of the hypothalamus. In contrast, theso-called satiety center is expressed in the ventromedial hypothalamusand is dominated by the leptin-regulated neuropeptide network.

Prolactin Releasing Peptides (SEQ ID NOS: 65-170)

Prolactin is produced by acidophilic pituitary lactotropes. Prolactinreleasing peptides act on lactotrope to release prolactin. PRL initiatesand maintains lactation in mammals, but normally only in mammary tissuethat has been primed with estrogenic sex hormones. A conjugated PRPcould be used to increase lactation in humans or animals.

Somatostatin (SEQ ID NOS: 171-201)

Also known as Growth Hormone Release Inhibiting Factors (GIF),somatostatin is a 14 amino acid peptide is secreted by both thehypothatamus and by d cells of the pancreas (its pancreatic version isdiscussed below). Somatostatin has been reported to modulatephysiological functions at various sites including pituitary, pancreas,gut and brain. It inhibits the release of growth hormone, insulin, andglucagon. It has many biological roles, including: inhibition of basaland stimulated hormone secretion from endocrine and exocrine cells, aneffect on locomotor activity and cognitive function, and possibletherapeutic value in small cell lung cancer. See Reubi, J. C. et al,Endocrinology, 110, 1049 (1982). A conjugated somatostatin could be usedto treat giantism in children or acromegaly in the adult.

Thyrotropin Releasing Hormone (THR) and Analogs (SEQ ID NOS: 202-214)

THR stimulates the production of thyroid stimulating hormone (TSH, alsoknown as thyrotropin) and prolactin secretion. In adults, TSH isresponsible for up-regulating general protein synthesis and inducing astate of positive nitrogen balance. In the embryo, it is necessary fornormal development. Hypothyroidism in the embryo is responsible forcretinism, which is characterized by multiple congenital defects andmental retardation. A conjugated THR could then be used as a therapeuticagent in the treatment of these disorders. It could also be used totreat pituitary causes of thyroid insufficiency or in the diagnosis ofhuman tumors of the thyroid.

C. Thyroid Hormones

Calcitonins (CT) & Caltitonins Precursor Peptides (SEQ ID NOS: 215-224)

Calcitonin (CT) is a 32-amino acid peptide secreted by C cells of thethyroid gland. Calcitonin is employed therapeutically to relieve thesymptoms of osteoporosis, although details of its mechanism of actionremain unclear. However, it has been observed that CT induces thesynthesis of parathyroid hormone (PTH) in isolated cells, which leads invivo to increased plasma Ca²⁺ levels. In addition, CT has been shown toreduce the synthesis of osteoporin (Opn), a protein made by osteoclastsand responsible for attaching osteoclasts to bone. Thus, usingconjugated CT as a therapeutic peptide would elevate plasma Ca²⁺ via PTHinduction and reduce bone reabsorption by decreasing osteoclast bindingto bone.

Calcitonins Gene Related Peptide (CGRP) (SEQ ID NOS: 225-253)

CGRP is a 37 amino acid peptide that results from alternative splicingof calcitonin gene transcripts. It exists in at least two forms:alpha-CGRP (or CGRP-I) and beta-CGRP (or CGRP-II). CGRP has considerablehomology with amylin and adrenomedullin, and is widely distributed bothcentrally and peripherally in organs including the skin, the heart, thepancreas, the lungs, and the kidneys. CGRP has many biological roles,affecting the nervous and cardiovascular systems, inflammation andmetabolism.

D. Parathyrold Hormones and Related Proteins

Parathyroid Hormones (PTH) (SEQ ID NOS: 254-293)

Parathyroid hormone (PTH) is synthesized and secreted by chief cells ofthe parathyroid in response to systemic Ca²⁺ levels. It plays a majorrole in the modulation of serum calcium concentration and thereby affectthe physiology of mineral and bone metabolism. The Ca²⁺ receptor of theparathyroid gland responds to Ca²⁺ by increasing intracellular levels ofPKC, Ca²⁺ and IP₃; this stage is followed, after a period of proteinsynthesis, by PTH secretion. The synthesis and secretion of PTH in chiefcells is constitutive, but Ca²⁺ regulates the level of PTH in chiefcells (and thus its secretion) by increasing the rate of PTH proteolysiswhen plasma Ca²⁺ levels rise and by decreasing the proteolysis of PTHwhen Ca²⁺ levels fall. The role of PTH is to regulate Ca²⁺ concentrationin extracellular fluids. The feedback loop that regulates PTH secretiontherefore involves the parathyroids, Ca²⁺, and the target tissuesdescribed below.

PTH acts by binding to cAMP-coupled plasma membrane receptors,initiating a cascade of reactions that culminates in the biologicalresponse. The body's response to PTH is complex but is aimed in alltissues at increasing Ca²⁺ levels in extracellular fluids. PTH inducesthe dissolution of bone by stimulating osteoclast activity, which leadsto elevated plasma Ca²⁺ and phosphate. In the kidney, PTH reduces renalCa²⁺ clearance by stimulating its reabsorption; at the same time, PTHreduces the reabsorption of phosphate and thereby increases itsclearance. Finally, PTH acts on the liver, kidney, and intestine tostimulate the production of the steroid hormone1,25-dihydroxycholecalciferol (calcitriol), which is responsible forCa²⁺ absorption in the intestine. A conjugated PTH could be used toregulate calcium homeostasis in patients with parathyroid hormonedeficiency states. Inhibitor analogues could be used to block PTH actionin renal failure or other patients with excessive PTH levels.

Parathyroid Hormone Related Proteins (PTHrP) (SEQ ID NOS: 294-309)

Parathyroid hormone-related protein (PTHrP) has received attention as aphysiological regulator attenuating chondrocytic differentiation andpreventing apoptotic cell death. PTHrP was initially identified as atumor-derived, secretory protein with structural similarity toparathyroid hormone (PTH), the major regulator of calcium homeostasis.PTH and PTHrP bind to a common G protein-coupled cell surface receptor(PTH/PTHrP or PTH-1 receptor) that recognizes the N-terminal (1-34)region of these peptides. Hence, when tumor-derived PTHrP enters thecirculation, it activates receptors in classic PTH target organs such asbone and kidney and elicits PTH-like bioactivity. By promoting boneresorption and inhibiting calcium excretion, circulating PTHrP givesrise to the common paraneoplastic syndrome of to malignancy-associatedhumoral hypercalcemia.

Although initially discovered in tumors, PTHrP was subsequently shown tobe expressed in a remarkable variety of normal tissues including thefetal and adult skeleton, where acting in concert with its aminoterminal PTH-1 receptor, it serves to regulate cellular growth anddifferentiation. The anabolic effects of intermittent PTH administrationon bone and its therapeutic potential in osteoporosis have beenextensively explored. With the recognition that PTHrP is the endogenousligand for the PTH/PTHrP receptor in osteoblasts, its use as an anabolicagent has also been investigated. Modified PTHrP peptides could be usedfor similar indications as PTH.

E. Pancreatic Hormones

The principal role of the pancreatic hormones is the regulation ofwhole-body energy metabolism, principally by regulating theconcentration and activity of numerous enzymes involved in catabolismand anabolism of the major cell energy supplies.

Amylin (SEQ ID NOS: 310-335)

Pancreatic beta-cell hormone amylin is a 37-amino-acid peptide relatedto CGRP and calcitonin. It is co-secreted with insulin from pancreaticbeta-cells. Amylin is deficient with type 1 diabetes mellitus. Amylinappears to work with insulin to regulate plasma glucose concentrationsin the bloodstream, suppressing the postherapeutic peptiderandialsecretion of glucagon and restraining the rate of gastric emptying.People with diabetes have a deficiency in the secretion of amylin thatparallels the deficiency in insulin secretion, resulting in an excessiveinflow of glucose into the bloodstream during the postherapeuticpeptiderandial period.

While insulin replacement therapy is a cornerstone of diabetestreatment, replacement of the function of both amylin and insulin mayallow a more complete restoration of the normal physiology of glucosecontrol. Type 2 diabetes is characterized by islet amyloid deposits,which are primarily composed of the amyloidogenic human form of isletamyloid polypeptide. A conjugated amylin could be used in the managementof diabetes to limit post prandial hyperglysemia.

Glucagon (SEQ ID NOS: 336-3761)

Glucagon is a 29-amino acid hormone synthesized by the a cells of theislets of Langerhans as a very much larger proglucagon molecule. Likeinsulin, glucagon lacks a plasma carrier protein, and like insulin itscirculating half life is also about 5 minutes. As a consequence of thelatter trait, the principal effect of glucagon is on the liver, which isthe first tissue perfused by blood containing pancreatic secretions.Glucagon binds to plasma membrane receptors and is coupled throughG-proteins to adenylate cyclase. The resultant increases in cAMP and PKAreverse all of the effects described above that insulin has on liver.The increases also lead to a marked elevation of circulating glucose,with the glucose being derived from liver gluconeogenesis and liverglycogenolysis. A conjugated glucagon construct could be used to managebrittle diabetes with recurrent hypoglycemia or to prevent or treatiatrogenic hypoglycemia.

Insulin and Insulin-Like Peptides (SEQ ID NOS: 377-382)

The earliest of these hormones recognized was insulin, a disulfidebonded dipeptide of 21 and 30 amino acids produced by the pancreas,whose major function is to counter the concerted action of a number ofhyperglycemia-generating hormones and to maintain low blood glucoselevels. Insulin is a member of a family of structurally and functionallysimilar molecules that include IGF-1, IGF-2, and relaxin. The tertiarystructure of all 4 molecules is similar, and all have growth-promotingactivities, but the dominant role of insulin is metabolic while thedominant roles of the IGFs and relaxin are in the regulation of cellgrowth and differentiation.

Insulin is synthesized as a preprohormone in the b cells of the isletsof Langerhans. Its signal peptide is removed in the cisternae of theendoplasmic reticulum and it is packaged into secretory vesicles in theGolgi, folded to its native structure, and locked in this conformationby the formation of 2 disulfide bonds. Specific protease activitycleaves the center third of the molecule, which dissociates as Cpeptide, leaving the amino terminal B peptide disulfide bonded to thecarboxy terminal A peptide.

Insulin generates its intracellular effects by binding to a plasmamembrane receptor, which is the same in all cells. The receptor is adisulfide-bonded glycoprotein. One function of insulin (aside from itsrole in signal transduction) is to increase glucose transport inextrahepatic tissue is by increasing the number of glucose transportmolecules in the plasma membrane. Glucose transporters are in acontinuous state of turnover. Increases in the plasma membrane contentof transporters stem from an increase in the rate of recruitment of newtransporters into the plasma membrane, deriving from a special pool ofpreformed transporters localized in the cytoplasm.

In addition to its role in regulating glucose metabolism (and itstherapeutic use in treating diabetes), insulin stimulates lipogenesis,diminishes lipolysis, and increases amino acid transport into cells.Insulin also modulates transcription, altering the cell content ofnumerous mRNAs. It stimulates growth, DNA synthesis, and cellreplication, effects that it holds in common with the IGFs and relaxin.A conjugated insulin could thus be used to manage diabetes.

NeuroPeptide Y (SEQ ID NOS: 383-389)

Neuropeptide Y (NPY), a peptide with 36 amino acid residues, is one ofthe most abundant neuropeptides in both the peripheral and the centralnervous systems. It belongs to the pancreatic polypeptide family ofpeptides. Like its relatives, peptide YY (PYY) and pancreaticpolypeptide (PP), NPY is bent into hairpin configuration that isimportant in bringing the free ends of the molecule together for bindingto the receptors.

NPY exerts a wide range of effects in the central nervous system (CNS)and the periphery. Its CNS actions include major effects on feeding andenergy expenditure, and alterations in heart rate, blood pressure,arousal and mood. In the periphery, NPY causes vasoconstriction andhypertension; it is also found in the gastrointestinal and urogenitaltract, implicating its functions by action upon gastrointestinal andrenal targets. In recent studies, hypothalamic NPY has been found toplay a fundamental role in developing the features of obesity, it is amajor transducer in the pathways signalling body fat to thehypothalamus, and in regulating body fat content. Leptin, an obese geneproduct, has been found to decrease NPY gene expression in obese (ob/ob)mice. Insulin and corticosteroids are also involved in the regulation ofhypothalamic NPY synthesis, with insulin decreasing and corticosteroidsincreasing NPY expression. A conjugated NPY could be used to treatobesity and MODM (Type II DM) in obese patients.

Pancreatic Polypeptides (PP) (SEQ ID NOS: 390-396)

Pancreatic polypeptide (PP) is a 36-amino acid hormone produced by Fcells within the pancreatic islets and the exocrine pancreas. It is amember of the PP fold family of regulatory peptides, and increasesglycogenolysis and regulates gastrointestinal activity. A conjugatedpancreatic polypeptide could thus be used to after absorption andmetabolism of foods.

Peptide YY (SEQ ID NOS: 397-400)

PYY is a thirty six amino acid long peptide, first isolated from porcineintestinal tissue and mainly localized in intestinal endocrine cells. Ithas many biological activities, including a range of activities withinthe digestive system and potent inhibition of intestinal electrolyte andfluid secretion.

Somatostatin (SEQ ID NOS: 171-201)

The somatostatin secreted by d cells of the pancreas is a 14-amino acidpeptide identical to somatostatin secreted by the hypothalamus. Inneural tissue somatostatin inhibits GH secretion and thus has systemiceffects. In the pancreas, somatostatin acts a paracrine inhibitor ofother pancreatic hormones and thus also has systemic effects. It hasbeen speculated that somatostatin secretion responds principally toblood glucose levels, increasing as blood glucose levels rise and thusleading to down-regulation of glucagon secretion. A conjugatedsomatostatin could then be used to aid in the management of diabetes.

F. Digestive Hormones

Cholecystokinin (CCK) & Related Peptides (SEQ ID NOS: 401-416)

CCK is a polypeptide of 33 amino acids originally isolated from pigsmall intestine that stimulates gallbladder contraction and bile flowand increases secretion of digestive enzymes from pancreas. It exists inmultiple forms, including CCK-4 and CCK-8, with the octapeptiderepresenting the dominant molecular species showing the greatestactivity. It belongs to the CCK/gastrin peptide family and isdistributed centrally in the nervous system and peripherally in thegastrointestinal system. It has many biological roles, includingstimulation of pancreatic secretion, gall bladder contraction andintestinal mobility in the GI tract as well as the possible mediation ofsatiety and painful stimuli. A conjugated CCK could be used indiagnostic studies of the gall bladder or in chronic cholecystisis.

Gastrin Releasing Peptide (GRP) (SEQ ID NOS: 417-429)

GRP is a 27-amino acid peptide originally isolated from porcinenon-antral gastric tissue, and is the homolog of the frog skin peptidenamed bombesin growth. It is widely distributed both centrally andperipherally in tissues including brain, lung and gastrointestinal tractIt regulates a variety of cell physiological processes includingsecretion, smooth muscle contraction, neurotransmission and cell growth.A conjugated GRP could be used in the treatment of adynamic ileus orconstipation in the elderly.

Gastrin & Related Peptides (SEQ ID NOS: 417-429)

Gastrin is a polypeptide of 17 amino acids produced by stomach antrum,which stimulates acid and pepsin secretion. Gastrin also stimulatespancreatic secretions. Multiple active products are generated from thegastrin precursor, and there are multiple control points in gastrinbiosynthesis. Biosynthetic precursors and intermediates (progastrin andGly-gastrins) are putative growth factors; their products, the amidatedgastrins, regulate epithelial cell proliferation, the differentiation ofacid-producing parietal cells and histamine-secretingenterochromaffin-like (ECL) cells, and the expression of genesassociated with histamine synthesis and storage in ECL cells, as well asacutely stimulating acid secretion. Gastrin also stimulates theproduction of members of the epidermal growth factor (EGF) family, whichin turn inhibit parietal cell function but stimulate the growth ofsurface epithelial cells. Plasma gastrin concentrations are elevated insubjects with Helicobacter pylori, who are known to have increased riskof duodenal ulcer disease and gastric cancer. The use of gastrin orgastrin antagonists as a therapeutic agent may therefore contribute totreating major upper gastrointestinal tract disease.

Gastrin Inhibitory Peptides (SEQ ID NOS: 417-429)

Gastrin inhibitory peptide is a polypeptide of 43 amino acids thatinhibits secretion of gastrin. A conjugated GIP could be used to treatsevere peptic ulcer disease.

Motilin (SEQ ID NOS: 430-433)

Motilin is a polypeptide of 22 amino acids that controlsgastrointestinal muscles. Motilin-producing cells are distributed in theduodenum, upper jejunum, and colorectal adenocarinomas and in midgutcarcinoids. Motilin stimulates gut motility.

Secretin (SEQ ID NOS: 434-441)

Secretin is a polypeptide of 27 amino acids secreted from duodenum at pHvalues below 4.5, stimulates pancreatic acinar cells to releasebicarbonate and H₂O. Secretin is a neurotransmitter (a chemicalmessenger) in the neuropeptide group. It is one of the hormones thatcontrols digestion (gastrin and cholecystokinin are the others). It is apolypeptide composed of 27 amino acids and is secreted by cells in thedigestive system when the stomach empties. Secretin stimulates thepancreas to emit digestive fluids that are rich in bicarbonate whichneutralizes the acidity of the intestines, the stomach to produce pepsin(an enzyme that aids digestion of protein), and the liver to producebile.

Secretin may be useful in treating autism. In one study, children withautistic spectrum disorders underwent upper gastrointestinal endoscopyand intravenous administration of secretin to stimulatepancreaticobiliary secretion. All three had an increasedpancreaticobiliary secretory response when compared with nonautisticpatients (7.5 to 10 mL/min versus 1 to 2 mL/min). Within 5 weeks of thesecretin infusion, a significant amelioration of the children'sgastrointestinal symptoms was observed, as was a dramatic improvement intheir behavior, manifested by improved eye contact, alertness, andexpansion of expressive language. These clinical observations suggest anassociation between gastrointestinal and brain function in patients withautistic behavior.

Vasoactive Intestinal Peptide (VIP) and Related Peptides (SEQ ID NOS:442-464)

VIP is a polypeptide of 28 residues produced by hypothalamus and GItract. It relaxes the GI, inhibits acid and pepsin secretion, acts as aneurotransmitter in peripheral autonomic nervous system, and increasessecretion of H₂0 and electrolytes from pancreas and gut. It wasoriginally discovered in lung and intestine and is also found in tissuesincluding brain, liver, pancreas, smooth muscle and lymphocytes. It isstructurally related to a family of peptides which include PACAP, PHI,secretin and glucagon. It has a diverse range of biological actionsincluding vasodilation, electrolyte secretion, modulation of immunefunction and neurotransmission. A conjugated VIP may be useful in thetreatment of achlorhydria, ischemic colitis and irritable bowel syndrome(IBS).

G. Natriuretic Peptides

There are three members in the natriuretic peptide hormone family,atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP, brainnatriuretic peptide), and C-type natriuretic peptide (CNP), that areinvolved in the regulation of blood pressure and fluid homeostasis.

Atrial-Natriuretic Peptides (ANP) (SEQ ID NOS: 465-507)

ANP is a 28-amino acid peptide hormone containing a disulfide bond. Itexerts natriuretic, diuretic, and vasorelaxant effects and play animportant role in the body's blood volume and blood pressurehomeostasis. See Smith, F. G. et al., J. Dev. Physiol. 12, 55 (1989).The mechanisms controlling ANP release have been the subject of intenseresearch, and are now fairly well understood. The major determinant ofANP secretion is myocyte stretch. Although much less is known about thefactors regulating BNP release from the heart, myocyte stretch has alsobeen reported to stimulate BNP release from both atria and ventricles.However, whether wall stretch acts directly or via factors such asendothelin-1, nitric oxide, or angiotensin II liberated in response todistension has not been established. Recent studies show that bystimulating endothelin type A receptors endothelin plays an importantphysiological role as a mediator of acute-volume load-induced ANPsecretion from atrial myocytes in conscious animals. In fact, endogenousparacrine/autocrine factors liberated in response to atrial wall stretchrather than direct stretch appears to be responsible for activation ofANP secretion in response to volume load, as evidenced by almostcomplete blockade of ANP secretion during combined inhibition ofendothelin type A/B and angiotensin II receptors. Furthermore, undercertain experimental conditions angiotensin II and nitric oxide may alsoexert a significant modulatory effect on stretch-activated ANPsecretion. The molecular mechanisms by which endothelin-1, angiotensinII, and nitric oxide synergistically regulate stretch-activated ANPrelease are yet unclear. Abstract Volume 75 Issue 11/12 (1997) pp876-885, Journal of Molecular Medicine. A conjugated would be useful inthe management of malignant hypertension or severe hypertension andrenal failure.

Brain Natriuretic Peptides (BNP) (SEQ ID NOS: 507-516)

Brain natriuretic peptide (BNP), a member of the natriuretic peptidefamily, is produced and released from cardiac ventricles. BNP regulatesthe body fluid volume, blood pressure, and vascular tones through theA-type guanylate cyclase-coupled receptor. The BNP plays a role inelectrolyte-fluid homeostasis such as atrial natriuretic peptides (ANP).A conjugated BNP could be useful in the management of heart failure.

C-Type Natriuretic Peptides (CNP) (SEQ ID NOS: 517-524)

C-type natriuretic peptide (CNP), the third member of the natriureticpeptide family, is produced in vascular endothelial cells (ECs) and actsas an endothelium-derived relaxing peptide. Although atrial and brainnatriuretic peptides are well known to be involved in the regulation ofcardiovascular and endocrine functions as circulating hormones, theroles of the C-type natriuretic peptide (CNP) remain unknown.

CNP is found principally in the central nervous system and vascularendothelial cells while ANP and BNP are cardiac hormones. ANP issynthesized mainly in the atria of the normal adult heart, while BNP isproduced by both the atria and ventricles.

H. Tachykinins (SEQ ID NOS: 525-627)

A family of peptides, including neurokinin A and substance P, that sharea common C terminal sequence (F-X-GLM-NH2) which is required for fullbiological activity including Neurokinin A, B, and Substance P.

Neurokinin A

Neurokinin A is a decapeptide, previously known as substance K. It is isa member of the tachykinin family of neuropeptides which includessubstance P and neurokinin B. It exhibits a variety of activitiesrelated to smooth muscle contraction, pain transmission,bronchoconstriction, vasodilation and modulation of the immune system.

Neuromedin

Neuromedins, smooth-muscle-stimulating peptides, are commonly dividedinto four groups: bombesin-like, kassinin-like, neurotensin-like andneuromedins U. These neuropeptides and their receptors are localized toall components of the HPA hypophyseal pituitary axis, the only exemptionseems to be neurokinin B, which is not detected in the adenohypophysis.Neuromedins exert a manifold effect on HPA axis, and their action on theadrenal suggests their involvement in the regulation of growth,structure and function of the adrenal cortex. Neuromedins may exert bothdirect and indirect effects on the adrenal cortex. Direct effect isproven by the stimulation of mineralo- and glucocorticoid therapeuticpeptides by isolated or cultured adrenocortical cells and bymobilisation of intracellular [Ca2+]. Indirect effects, on the otherhand, may be mediated by ACTH, arginine-vasopressin, angiotensin II,catecholamines or by other regulatory substances of medullary origin.

Substance P and Related Peptides

Substance P is an eleven amino acid peptide, first isolated from brainand intestine. It has been proposed as a neuromodulator involved in paintransmission in the spinal cord. It also affects contraction of smoothmuscle, reduction of blood pressure, stimulation of secretory tissue,and release of histimine from mast cells.

I. Renin Related Peptides

Angiotensins (SEQ ID NOS: 628-677)

Angiotensin is a 10 amino acid peptide derived from enzymatic cleavageof a2-globin by the kidney enzyme renin. The C-terminal 2 amino acidsare then released to yield angiotensin I, which is responsible foressential hypertension through stimulated synthesis and release ofaldosterone from adrenal cells. It is a multifunctional hormoneregulating blood pressure, plasma volume, neuronal function thirst, andwater intake.

Angiotensin II is an octapeptide derived from angiotensin I byangiotensin converting enzyme, and is widely distributed both centrallyand peripherally in organs such as the heart, the kidneys, and theliver. Angiotensin IV is the terminal hexapeptide fragment ofangiotensin II formed metabolically by proteolytic cleavage from eitherangiotensin I or angiotensin II. It plays a role in vascular control,cardiac growth, renal blood flow and memory function.

Angiotensin II is the key peptide hormone that regulates vascular smoothmuscle tone, blood pressure, free water intake and sodium retension. Itcontrols vascular homeostasis compensating for loss of intravascularvolume by stimulating increased vasospastic tone, increase sodiumretention and increased free water intake.

Renin Substrates and Inhibitors (SEQ ID NOS: 678-684)

Renin is a very specific aspartic protease, which is synthesized andreleased by differentiated smooth muscle cells in the vasculature of thekidney called granular epithelial cells. Renin is specific for itssubstrate, angiotensinogen, which it cleaves specifically at theLeu¹⁰-Val¹¹ bond to form the decapeptide, angiotensin I (AI). Therenin-angiotensin system is involved in the control of fluid and mineralbalance throughout the vertebrates. Renin can be found in mammals,birds, reptiles, amphibians, bony fishes, cartilaginous fishes, andagnathans. Specific renin inhibitors can also be designed, withtherapeutic applications for treatment of for example hypertension andcongestive heart failure (Blundell et al., 1987).

J. Endothelins and Related Peptides (SEQ ID NOS: 685-744)

The endothelin peptide family consists of the 21 amino acid isoformsendothelin-1, endothelin-2, endothelin-3, sarafotoxin (a snake venom)and scorpion toxin.

Endothelins (ET) and Big Endothelins

Endothelins are found on endothelial cells in a wide variety of organsystems. Examples of pathologies and physiological processes associatedwith changes in endothelin levels and synthetics include:atherosclerosis and hypertension, coronary vasospasm, acute renalfailure, changes in intracellular Ca2⁺ levels, and effects on therenin-angiotensin system. Endothelins are released in reponse tovariations in angiotensin II, vasopressin, and cytokines (e.g. TGF-βmTNF-α, IL-β-) levels as well as other physiological events includingincrease blood flow.

The endothelin family of peptides consists of highly potent endogenousvasoconstrictor agents first isolated from endothelial cell supernatant.They regulate blood flow to organs by exporting a vasoconstrictiveeffect on arteries. Endothelins are derived from big-endothelin, whichis cleaved by a unique membrane-bound metalloprotease,endothelin-converting enzyme, into the 21-amino-acid bioactive forms(ET-1, ET-2 and ET-3).

Of the 3 isoforms (ET-1, ET-2, ET-3), endothelin-1 is the major isoformand plays an important role for regulation of vascular function.Endogenous endothelin peptides and their receptors are differentiallydistributed throughout the many smooth muscle tissues including bloodvessels, uterus, bladder and intestine. Through this widespreaddistribution and localization, they exert biological functions inregulating vascular tone and causing mitogenesis. ETs and their receptorsubtypes are also present in various endocrine organs. It appears to actas a modulator of secretion of prolactin, gonadotropins GH and TSH.Endothelin may also be the disease marker or an etiologic factor inischemic heart disease, atherosclerosis, congestive heart failure, renalfailure, systemic hypertension, pulmonary hypertension, cerebralvasospasm.

Exogenously administered endothelin-1 has been demonstrated to increaseperipheral resistance and blood pressure in a dose-dependent manner.However, during the first minutes of intravenous administrationendothelins also decrease peripheral resistance and blood pressure,presumably due to the release of vasodilatory compounds such as nitricoxide, prostacyclin, and atrial natriuretic peptide.

ET(A) Receptor Antagonists

Endothelin receptors exist as two types: A (ET-A) and B (ET-B1 andET-B2). ET-A receptors are responsible for while ET-B1 and ET-B2 mediatevasorelaxation and vasoconstriction respectively.

Sarafotoxln Peptides

As already described, endothelin (ET) peptides are potent growth factorsbinding to G protein-coupled receptors. Sarafotoxins (S6) isolated fromAtractaspis engaddensis are highly homologous to endothelins.Sarafotoxin peptides have marked vasoconstrictive activity and areresponsible for the ischemic limb loss that follows snake or scorpionbites. They could be used therapeutically as a peptidase stabalizedpeptide as a vasopressive agent in shock and sepsis.

K. Opioid Peptides (SEQ ID NOS: 745-927)

Opioids are a large class of drugs, used clinically as painkillers, thatinclude both plant-derived and synthetic alkaloids and peptides foundendogenously in the mammalian brain. While the plant-derived alkaloidshave been known and used for thousands of years, the endogenous opioidpeptides were discovered only in the mid-1970s.

Opioids include casomorphin peptides, demorphins, endorphins,enkephalins, deltorphins, dynorphins, and analogs and derivatives ofthese.

Casomorghin Peptides

Casomorphin peptides are novel opioid peptides derived fromcasein(§-casomorphins). Beta casomorphins are the more exstensivestudied opioid peptides arising from food proteins (beta-caseins). Theywere originally isolated from bovine beta-casein, the same sequencesoccur in ovine and buffalo beta-caseins.

Dermorphins

Demorphin is a is a seven amino acid peptide, originally isolated fromPhylomedusa sauvagel frog skin. It is a ligand which binds with highaffinity to the μ opioid receptor, and has many biological rolesincluding analgesia, endocrine modulation, immunomodulation, increasedK⁺ conductance and inhibition of action potentials.

Dynorphin/New-Endorphin Precursor Related Peptides

Dynorphins are a class of endogenous opioids that exist in multipleforms in the central nervous system. Dynorphins are derived from theprecursor prodynorphin (proenkephalin B). Dynorphin, also known asDynorphin A1-17, is a well known opioid which has the sequenceTyr-Gly-Gly-Phe-Leu⁵-Arg-Arg-Ile-Arg-Pro¹⁰-Lys-Leu-Lys-Trp-Asp¹⁵-Asn-Gln.SEQ ID NO:1. A number of derivatives and analogs of dynorphin are knownincluding Dyn A1-13, SEQ ID NO: 2 Dyn A2-13, SEQ ID NO:3, Dyn A1-12, DynA2-12 and Dyn A2-17 as well as amide analogs such as those mentioned inU.S. Pat. No. 4,462,941 of Lee et al., N-terminus truncated dynorphinanalogs such as those described in International Patent Application WO96/06626 of Lee et al. and des-Tyr or des-Tyr-Gly analogs such as thosedisclosed in International Patent Application WO 93/25217 also of Lee etal. The dynorphis are highly potent opioids, and demonstrate selectiveaffinity for the kappa receptor. See Goldstein, A., Peptides, Structureand Function, Proceedings of the 8th American Peptides Symposiym, Hruby,V. J. and Rich, D. H., eds., 409 (1983).

Endorphins

The endorphis are derived from the precursor protein-lipotropin. Theyhave been found to elicit several biological reactions such asanalgesia, behavioral changes and growth hormone release. See Akil, H.et al., Ann. Rev. Neurosci., 7, 223 (1984).

Enkepalins & Related Peptides

Enkephalins and endorphins are neurohormones that inhibit transmissionof pain impulses. The activity of neurons in both the central andperipheral nervous systems is affected by a large number ofneurohormones that act on cells quite distant from their site ofrelease. Neurohormones can modify the ability of nerve cells to respondto synaptic neurotransmitters. Several small peptides with profoundeffects on the nervous system have been discovered recently, for exampleenkephalins (e.g. Met-enkephalin and Leu-enkephalin) and endorphins(e.g. β endorphin). These three contain a common tetrapeptide sequence(Tyr-Gly-Gly-Phe) that is essential to their functions. Enkephalins andendorphins function as natural pain killers or opiates and decrease thepain responses in the central nervous system. See also Akil, H. et al.,Ann. Rev. Neurosci., 7, 223 (1984).

L. Thymic Peptides (SEQ ID NOS: 928-934)

The thymus is thought to be responsible for the development andregulation of T cell immunity in both infants and adults. The thymusseems to exert its regulatory functions through the secretion of variousnoncellular, hormonelike products via its epithelial cells, calledthymic peptides.

Thymic peptides are reported to have many effects on T cells. Severalstudies have reported that thymic peptides can assist development ofimmature, precursor cells into fully competent T cells. Thymic peptidesseem to regulate the expression of various cytokine and monokinereceptors on T cells and induce secretion of IL-2, interferon alpha, andinterferon gamma (disease-fighting substances) when the immune system ischallenged. There are reports that the use of thymic hormones inchildren with immuno-deficiencies caused by chemotherapy has resulted inan increase in circulating T cells, normalization of T cell subsets, andrestoration of delayed hypersensitivity reactions.

Thymopoietin

Thymopoietin is the largest of the known thymic hormones and consists of49 amino acids.

Thymulin

Previously known as thymic serum factor, thymulin is the smallest of thechemically characterized thymic hormones and consists of 9 amino acids.Thymulin is the hormone responsible for stimulating the production ofimmune-system T cells

Thymopentin

Thymopentin is a small, synthesized thymic peptide drug, also known astherapeutic peptide-5 or Timunox. In the U.S. it is being developed asan AIDS therapy by the Immunobiology Research Institute. Thymopentin hasbeen studied more extensively than most other thymic peptide drugs. Atleast one study has claimed a significant rise in T cells and slightclinical improvement in those patients who received thymopentin threetimes a week, compared to untreated control participants. Compared tothe 14 untreated control participants, those taking the drug showedgreater “immunologic stability” and some clinical improvement.

Thymosin

Thymosin is a mixture of 15 or more proteins. One of these proteins isthymosin alpha-1 which consists of 28 amino acids. Thymosin hastherapeutic use for the treatment of primary immunodeficiencies and as abooster for influenza vaccine in renal dialysis patients. It is alsobeing tested in ongoing clinical trials for activity against chronichepatitis B and C, HIV infection, and certain forms of cancer.

Thymic Humoral Factor (THF)

THF is a thymic peptide currently being examined as an anti-HIVtreatment. In preclinical studies in rats with CMV-relatedimmunosuppression, THF restored immune competence through modulation ofT cells. In addition, it may have therapeutic use in the treatment ofherpes, causing (at least in one study) the viral infection's rapidregression and increase of T-cell populations.

L. Other Peptides

Adrenomedullin Peptides (AM) (SEQ ID NOS: 935-945)

Adrenomedullin is a potent vasodilator peptide that exerts major effectson cardiovascular functions. Its systemic administration causes a rapidand profound fall in blood pressure and an increase in pulmonary bloodflow. Its other actions are bronchodilatation, being an inhibitor ofdrinking behavior and an inhibitor of angiotensin-induced aldosteronesecretion. See The Journal of Biological Chemistry, Vol. 270, No. 43, pp25344-25347, 1995 and in the references cited therein

Allatostatin Peptides (SEQ ID NOS: 946-949)

Allatostatins are 6-18 amino acid peptides synthesized by insects tocontrol production of juvenile hormones, which in turn regulatefunctions including metamorphosis and egg production. Whileneuropeptides of the allatostatin family inhibit in vitro production ofjuvenile hormone, which modulates aspects of development andreproduction in the cockroach, Diploptera punctata, they are susceptibleto inactivation by peptidases in the hemolymph, gut, and bound tointernal tissues.

Amyloid Beta-Protein Fragments (Aβ Fragments) (SEQ ID NOS: 950-1010)

These are the principle component of the amyloid plaques that accumulateintracellularly and extracellularly in the neuritic plaques in the brainin Alzheimer's Disease. Aβ is a 4.5 kD protein, about 40-42 amino acidslong, that is derived from the C-terminus of amyloid precursor protein(APP). APP is a membrane-spanning glycoprotein that, in the normalprocessing pathway, is cleaved inside the Aβ protein to produce α-sAPP,a secreted form of APP. Formation of α-sAPP precludes formation of Aβ.It has been proposed that Aβ accumulates by virtue of abnormalprocessing of APP, so that compounds that inhibit the activity ofenzymes responsible for Aβ production are being sought. See, e.g.,Wagner et al. Biotech. Report (1994/1995), pp. 106-107; and Selkoe(1993) TINS 16:403-409. Under certain conditions Aβ peptides firstaggregate and then are deposited as a folded β-sheet structure that ischaracteristic of amyloid fibrils. β-amyloid (1-42) forms aggregates ata significantly greater rate and to a greater extent than β-amyloid(1-40).

Antimicrobial Peptides (SEQ ID NOS: 1011-1047)

Antimicrobial peptides are a key component of the innate immune systemsof most multicellular organisms, being active against one or moremicroorganisms such as bacteria, fungi, protozoa, yeast, andmycobacteria. Examples of such peptides include defensin, cecropin,buforin, and magainin. Despite broad divergences in sequence andtaxonomy, most antimicrobial peptides share a common mechanism ofaction, i.e. membrane permeabilization of the pathogen. They areclassified in two broad groups: linear and cyclic. In the linearantimicrobial peptides, there are two subgroups: linear peptides tendingto adopt α-helical amphipathic conformation and linear peptides ofunusual composition, rich in amino acids such as Pro, Arg, or Trp. Thecyclic group encompasses all cysteine-containing peptides, and can befurther divided into two subgroups corresponding to single or multipledisulfide structures.

Most antimicrobial peptides provoke an increase in plasma membranepermeability. There is also evidence of other mechanisms, such asinhibition of specific membrane proteins, synthesis of stress proteins,arrest of DNA synthesis, breakage of single-strand DNA by defensins,interaction with DNA (without arrest of synthesis) by buforins, orproduction of hydrogen peroxide. Antimicrobial peptides can also act bytriggering self-destructive mechanisms such as apoptosis in eukaryoticcells or autolysis in bacterial targets. Antimicrobial peptides are alsoknown to act as inhibitors of enzymes produced by pathogenic organisms,either by serving as pseudo-substrates or by tight binding to the activesight that disturbs the access of the substrate.

Increased levels of antimicrobial peptides have been reported forseveral animal and human infections for example for α-defensins inMycobacterium, Pasteurella, or Cryptoporidium infections and for avariety of peptides in blisters and wound fluid. Inflammatory situationsor stimuli are also associated with induction of antibiotic peptides.

Depleted levels of antimicrobial peptides are associated to severalpathologies. Thus, patients of specific granule-deficiency syndrome,completely lacking in α-defensins, suffer from frequent and severebacterial infections. Low levels of histatins from saliva in HIVpatients has been correlated with a higher incidence of oral candidiasisand fungal infections. Perhaps the most compelling illustration of theimplication of antimicrobial peptides in human pathology comes fromcystic fibrosis, a genetic disease associated with recurrent bacterialinfections of the airways. The defective chloride channel causing thedisease increases the salinity of the alveolar fluid, and thus impairsthe bactericidal activity of β-defensins, which are salt sensitive.Andreu D, (Ed.)(1998) “Antimicrobial peptides” Biopolymers (PeptideScience) vol 47, N^(o) 6, pp413-491. A. Andreu, L. Rivas (1998) AnimalAntimicrobial Peptides: An Overview, Biopolymers (Pep. Sci.) 47:pp415-433.

Antioxidant Peptides (SEQ ID NOS: 1048-1050)

Antioxidants are agents that prevents oxidative damage to tissue.Mammalian cells are continuously exposed to activated oxygen speciessuch as superoxide, hydrogen peroxide, hydroxyl radical, and singletoxygen. These reactive oxygen intermediates are generated in vivo bycells in response to aerobic metabolism, catabolism of drugs and otherxenobiotics, ultraviolet and x-ray radiation, and the respiratory burstof phagocytic cells (such as white blood cells) to kill invadingbacteria such as those introduced through wounds. Hydrogen peroxide, forexample, is produced during respiration of most living organismsespecially by stressed and injured cells.

One example of antioxidant peptides is natural killer-enhancing factor B(NKEF-B), which belongs to a highly conserved family of recentlydiscovered antioxidants. Natural killer-enhancing factor (NKEF) wasidentified and cloned on the basis of its ability to increase NKcytotoxicity. Two genes, NKEF-A and -B, encode NKEF proteins andsequence analysis presented suggests that each belongs to a highlyconserved family of antioxidants. The role of NKEF-B as an antioxidanthas been demonstrated by its protection of transfected cells tooxidative damage by hydrogen peroxide. NKEF-B has antioxidant activitiestoward prooxidants such as alkyl hydroperoxide and MeHg. Together withits antioxidant activity, the induction of NKEF-B by HP indicates thatNKEF-B is an important oxidative stress protein providing protectionagainst a variety of xenobiotic toxic agents.

Apoptosis Related Peptides (SEQ ID NOS: 1051-1075)

Animal cells can self-destruct via an intrinsic program of cell death(Steller, 1995). Apoptosis is a form of programmed cell death that ischaracterized by specific morphologic and biochemical properties (Wyllieet al., 1980). Morphologically, apoptosis is characterized by a seriesof structural changes in dying cells: blebbing (i.e. blistering) of theplasma membrane, condensation of the cytoplasm and nucleus, and cellularfragmentation into membrane apoptotic bodies (Steller, 1995; Wyllie etal., 1980).

Biochemically, apoptosis is characterized by the degradation ofchromatin, initially into large fragments of 50-300 kilobases andsubsequently into smaller fragments that are monomers and multimers of200 bases (Oberhammer et al., 1993; Wyllie, 1980). Other biochemicalindicators of apoptosis are induced or increased levels of the proteinclusterin (Pearse et al., 1992), also known as TRPM-2 or SGP-2, andactivation of the enzyme typell transglutaminase, which crosslinksproteins to the envelope of apoptotc bodies (Fesus et al., 1991).Apoptosis is a complex phenomenon of related morphological andbiochemical processes that can vary with tissue and cell type (Zakeri etal., 1995).

The execution of apoptosis minimizes the leakage of cellularconstituents from dying cells (apoptosis causes the cell to involute).For example, proteases could damage adjacent cells or stimulate aninflammatory response. This cardinal feature of apoptosis distinguishesit from necrosis, which usually results from trauma that causes injuredcells to swell and lyse, releasing the cytoplasmic material thatstimulates an inflammatory response (Steller, 1995; Wyllie et al., 1980)

Bag Cell Peptides (BCP) (SEQ ID NOS: 1076-1080)

The neuropeptidergic bag cells of the marine mollusc Aplysia californicaare involved in the egg-laying behavior of the animal. Theseneurosecretory cells synthesize an egg-laying hormone (ELH) precursorprotein, yielding multiple bioactive peptides, including ELH, severalbag cell peptides (BCP) and acidic peptide (AP). The bag cells of themarine mollusc Aplysia californica are well-characterized neuroendocrinecells that initiate egg laying. During sexual maturation, these cells(bag cell neurons), develop the capability of storing hormones that arereleased during periods of nervous system stimulation. The hormones areimportant to the process of egg laying, and so must not be releasedbefore the animal is sexually mature. Alpha-bag cell peptide belong to asmall family of structurally related peptides that can elicit bag-cellactivity in vitro.

Bombesin (SEQ ID NOS: 1081-1090)

Bombesin is a bioactive tetradecapeptide neuropeptide that belongs to afamily of peptides sharing a common C terminal sequence,Trp-Ala-X-Gly-His-Met-NH2, and the N terminal region. It has amodulatory role found in nerves of the brain and gut that preventsgastric injury by release of endogenous gastrin. The mammalian homologueof bombesin is gastrin-releasing peptide (GRP).

Bone Gla Protein Peptides (SEQ ID NOS: 1091-1097)

Osteocalcin (bone Gla-protein, or BGP) is produced and secreted byosteoblasts in the process of bone formation. As with collagen, thisprotein is a component of bone matrix. Serum osteocalcin rises when boneformation rates increase. Levels are high during puberty when bonegrowth is most rapid. Often levels are also high in diseases having highbone turnover, such as hyperparathyroidism and hyperthyroidism. Inpostmenopausal osteoporosis, osteocalcin levels are sometimes increased,reflecting the increased turnover of bone secondary to rapid boneresorption. In senile osteoporosis, occurring in more elderly subjects,osteocalcin levels are more likely to be low, reflecting reduced ratesof both bone turnover and bone formation. A treatment regimen thatincreases bone formation also raises the serum osteocalcin levels.

CART Peptides (SEQ ID NOS: 1098-1100)

Cocaine and amphetamine regulated transcript peptide (CART), is arecently discovered hypothalamic peptide with a potent appetitesuppressing activity. In the rat the CART gene encodes a peptide ofeither 129 or 116 amino acid residues whereas only the short form existsin humans. The predicted signal sequence is 27 amino acid residuesresulting in a prohormone of 102 or 89 residues. The C-terminal end ofCART, consisting of 48 amino acid residues and 3 disulphide bonds, isthought to constitute a biologically active part of the molecule.

In the central nervous system CART is highly expressed in manyhypothalamic nuclei, some of which are involved in regulating feedingbehavior. The CART mRNA is regulated by leptin, and the expressed CARTis a potent inhibitor of feeding that even overrides the feedingresponse induced by neuropeptide Y. The putative CART receptor istherefore a potential therapeutic target for an anti-obesity drug. SeeCART, a new anorectic peptide Thim L; Kristensen P; Larsen P J; Wulff BS, Int J Biochem Cell Biol, 30(12):1281-4 1998 December

Cell Adhesion Peptides (SEQ ID NO: 1101)

Cellular adhesion peptides are directly involved in the cellularresponse to external stimuli. For example, during an inflammatoryresponse, leukocytes must leave the plasma compartment and migrate tothe point of antigenic insult. The mechanism of this migratory event isa complex interplay between soluble mediators and membrane-boundcellular adhesion molecules. Soluble cellular chemotactic factors, whichare produced in the damaged tissue by a variety of resident cells, setup a chemical concentration gradient out to the plasma compartment.Interaction of these factors with their receptors on leukocytes leads toa directional migration of the leukocytes toward increasingconcentrations of the chemotactic factor. Simultaneously, variousadhesion peptides are upregulated on the leukocyte which mediate theinitial rolling on the endothelial tissue, binding to a specific ligandon the activated endothelial tissue, and finally migration betweenendothelial cells into the tissue. The steps in this cascade of eventsare mediated by the interaction of specific cell surface proteins,termed “cell adhesion molecules such as, E-selectin (ELAM-1, endothelialleukocyte adhesion molecule-1), ICAM-1 (intercellular adhesionmolecule-1), and VCAM-1 (vascular cell adhesion molecule-1).

Chemotactic Peptides (SEQ ID NOS: 1102-1113)

Chemotactic peptides are peptides that stimulate the migration of whitecells, leukocytes and macrophages into tissues at the site of infectionor injury or alternatively the prevent the migration of these same cellsaway from these sites.

Complement Inhibitors (SEQ ID NOS: 1114-1120)

Inhibition of complement attack on xenotransplants may be accomplishedby the use of complement inhibitors. The rejection of transplantedorgans may involve both an extremely rapid hyperacute rejection (HAR)phase and a slower cellular rejection phase. HAR of xenotransplants isinitiated by preformed “natural” antibodies that bind to donor organendothelium and activate complement attack by the recipient immunesystem. Activation of complement leads to the generation of fluid phase(C3a, C5a) and membrane bound (C3b and C5b-9, i.e., C5b, C6, C7, C8, andC9) proteins with chemotactic, procoagulant, proinflammatory, adhesive,and cytolytic properties. Complement inhibitors inhibit this process.

Cortistatin Peptides (SEQ ID NOS: 1121-1124)

Cortistatin, whose mRNA accumulates during sleep deprivation, apparentlyacts by antagonizing the effects of acetylcholine on corticalexcitability, thereby causing synchronization brain slow waves.Cortistatin-14 (CST-14) shares 11 of its 14 residues withsomatostafin-14 (SRIF-14), yet its effects on sleep physiology,locomotor behavior and hippocampal function are quite different fromthose of somatostatin.

Fibronectin Fragments & Fibrin Related Peptides (SEQ ID NOS: 1125-1174)

Fibronectin is a large glycoprotein that is composed of blocks of threetypes of repeating, homologous peptide sequences. Several of thehomologous blocks form functional domains that are organized in a lineararray on two nearly identical subunit arms. Each arm can be divided intofunctional domains, which are often referred to by one of the substanceswhich bind in that region, for example the heparin-binding fragment, thefibrin binding fragment, and the cell-binding fragment. In several celltypes, the Arg-Gly-Asp (RGD) sequence in the cell-binding domain offibronectin interacts with a cell-surface glycoprotein designatedIib/IIIa. Fibronectin also binds to extracellular and basement-membranecomponents, to the envelope glycoprotein of viruses, to a variety ofbacteria including staphylococci and streptococci, and to parasites suchas Trypanosoma cruzi and Leishmania species.

Fibronectin has several adhesive functions, for example cell-to-celladhesion, cell-to-basement-membrane attachment, and clot stabilization.In addition, fibronectin promotes embryogenesis, nerve regeneration,fibroblast migration, macrophage function, and pathogen (virus, fungus,bacteria, and protozoa) binding to mammalian cells and extracellularmatrix. Thus, fibronectin is involved in the pathogenesis of infectionsfrom the initiation of the infection through the final stages of woundhealing. See Proctor, R. A., Rev. Infect. Dis., 9, 317 (1987).

FMRF and Analog Peptides (SEQ ID NOS: 1175-1187)

FMRF are neuropeptides encoded in the FMRF amide gene and have a commonC-terminal FMRFamide but different N-terminal extensions.FMRFamide-related peptides (FaRPs) are present throughout the animalkingdom and affect both neural and gastrointestinal functions. Organismshave several genes encoding numerous FaRPs with a common C-terminalstructure but different N-terminal amino acid extensions.

Galanin & Related Peptides (SEQ ID NOS: 1188-1208)

Galanin is a 29-30 amino acid peptide originally isolated from pig smallintestine. It is found in two biologically active forms: GAL (1-19), andGAL (1-30), a non-amidated form. It has many biological roles including:the inhibition of the release of biogenic amines in the hypothalamus,the pre- and post-synaptic inhibition of cholinergic function, themaintenance of gastrointestinal homeostasis, and the regulation ofinsulin and glucagon secretion.

Growth Factors & Related Peptides (SEQ ID NOS: 1209-1240)

Growth factors are a family of proteins that regulate cell division.Some growth factors are cell type specific, stimulating division of onlythose cells with appropriate receptors. Other growth factors are moregeneral in their effects. There are also extracellular factors thatantagonize the effects of growth factors, slowing or preventing division(for example transforming growth factor beta and tumor necrosis factor).These extracellular signals act through cell surface receptors verysimilar to those for hormones, and by similar mechanisms: the productionof intracellular second messangers, protein phosphorylation, andultimately, alteration of gene expression.

Gtherapeutic Peptide-Binding Protein Fragments (SEQ ID NOS: 1241-1246)

Members of a family of Gtherapeutic peptide-binding regulatory proteins(G-proteins) transduce signals from membrane-bound receptors tointracellular effectors. The family includes G_(s) and G_(i), which areresponsible for simulation and inhibition, respectively, of adenylatecyclase. Transducin (T), localized in the disc membranes of retinal rodouter segments, couples activation of rhodopsin by light to increasedcyclinc GMP phosphodiesterase activity. G_(o), found originally inbovine brain, is a fourth member of the family.

Purified G proteins have similar physical properties. They areheterodimers composed of α, β, and γ subunits. The α subunits bind andhydrolyze Gtherapeutic peptide. See S. M. Mumby et al., PNAS 83, 265(1986) and Lehninger p. 764.

Guanylin and Uroguanylin (SEQ ID NOS: 1247-1249)

Guanylin and uroguanylin are peptides isolated from intestinal mucosa,and urine, which regulate cyclic GMP production in enterocytes bind toand activate guanylate cyclase C and control salt and water transport inmany epithelia in vertebrates, mimicking the action of severalheat-stable bacteria enterotoxins. In the kidney, both of them havewell-documented natriuretic and kaliuretic effects.

Chloride secretion in the intestine is regulated by these hormones viaactivation of guanylate cyclase C (GC-C). Both peptides are expressed ina variety of tissues and organs, including the kidney. In the isolatedperfused kidney and in vivo these hormones induce natriuresis anddiuresis, however, localisation and cellular mechanisms of their actionin the kidney are still unknown.

Inhibin Peptides (SEQ ID NOS: 1250-1255)

Inhibin is composed of two subunits (α is 134 amino acids; β is 115 and116 amino acids). Its role is inhibition of FSH secretion. The twoinhibin isoforms, inhibin A and inhibin B, are produced by the gonads inthe course of gamete maturation and have different patterns of secretionduring the menstrual cycle. Inhibins are also produced by the placentaand fetal membranes and may be involved in physiological adaptation ofpregnancy. Clinically, inhibins may serve as sensitive tumor markers inpostmenopausal women, or as useful tools for evaluating ovarian reservein infertile women; they may also be used in the diagnosis ofmaterno-fetal disorders and to prevent maturation of the ovum or toinhibit ovulation.

Interleukin (IL) and Interleukin Receptor Proteins (SEQ ID NOS:1256-1263)

Interleukins are growth factors targeted to cells of hematopoieticorigin. A variety of biological activities associated with immune andinflammatory responses have been ascribed to interleukins. Theseresponses include fever, cartilage breakdown, bone resorption, thymocyteproliferation, activation of T and B lymphocytes, induction ofacute-phase protein synthesis from hepatocytes, fibroblastproliferation, and differentiation and proliferation of bone marrowcells.

Laminin Fragments (SEQ ID NOS: 1264-1284)

Laminin, the major noncollagenous glycoprotein of basement membranes,has been shown to promote the adhesion, spreading, and migration of avariety of tumor cell types in vitro. In particular, the major currentstudies in the laboratory utilize intact laminin, purified proteolyticfragments of laminin, and synthetic peptides of laminin to identifyfunctionally active sites on this large protein. Components of suchbasement membranes are important modulators of growth, development, anddifferentiation for various cell types. A conjugated laminin could beused to prevent inflamation or fibrosis in tissues.

This category also includes the peptide kringle-5 (or K-5). As usedherein, the term “kringle 5” refers to the region of mammalianplasminogen having three disulfide bonds which contribute to thespecific three-dimensional confirmation defined by the fifth kringleregion of the mammalian plasminogen molecule. One such disulfide bondlinks the cysteine residues located at amino acid positions 462 and 541,a second links the cysteine residues located at amino acid positions 483and 524 and a third links the cysteine residues located at amino acidpositions 512 and 536. The term “kringle 5 peptide peptides” refers topeptides with anti-angiogenic activity of between 4 and 104 amino acids(inclusive) with a substantial sequence homology to the correspondingpeptide fragment of mammalian plasminogen.

Leptin Fragment Peptides (SEQ ID NOS: 1285-1281)

Leptin, the protein product of the obesity gene, is secreted by fatcells. Leptin is involved in the regulation of bodyweight and metabolismin man and might also be involved in the pathophysiology of the insulinresistance syndrome, which is associated with the development ofcardiovascular diseases

Leucokinins (SEQ ID NOS: 1289-98)

Leucokinins are a group of widespread insect hormones that stimulate gutmotility and tubule fluid secretion rates. In tubules, their majoraction is to raise chloride permeability by binding to a receptor on thebasolateral membrane.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) (SEQ ID NOS:1299-1311)

It is a thirty-eight amino acid peptide first isolated from ovinehypothalamus, which also occurs in a 27 amino acid form called PACAP-27.PACAP has been localized in the hypothalamus, elsewhere in the brain,respiratory tract and gastrointestinal system. It has many biologicalactions, including neurotransmitter and hormonal functions, involvementin regulation of energy metabolism, and neuronal cytoprotectiveactivity.

Pancreastatin (SEQ ID NOS: 1312-1324)

Pancreastatin is a 49 amino acid peptide first isolated, purified andcharacterized from porcine pancreas. Its biological activity indifferent tissues can be assigned to the C-terminal part of themolecule. Pancreastatin has a prohormonal precursor, chromogranin A,which is a glycoprotein present in neuroendocrine cells, including theendocrine pancreas

Polypeptides (SEQ ID NOS: 1325-1326)

These are repetitive chains. Two examples are provided:(pro-Hyp-Gly)10*20H20 and Poly-L-Lysine Hydrochloride.

Signal Transduction Reagents (SEQ ID NOS: 1327-1367)

Signal transduction is the process by which an extracellular signal (forexample chemical, mechanical, or electrical) is amplified and convertedto a cellular response. Many reagents are involved in this process, forexample achatin-1, glycogen synthase, autocamtide 2, calcineurinautoinhibitory peptide, calmodulin dependent protein kinase II,calmodulin dependent protein kinase substrate, calmodulin dependentprotein kinase substrate analog, CKS-17, Cys-Kemptide, autocamtide 2,malantide, melittin, phosphate acceptor peptide, protein kinase Cfragments, P34cd2 kinase fragment, P60c-src substrate II, protein kinaseA fragments, tyrosine protein kinase substrate, syntide 2, S6 kinasesubstrate peptide 32, tyrosine specific protein kinase inhibitor, andtheir derivatives and fragments.

Thrombin Inhibitors (SEQ ID NOS: 1368-1377)

Thrombin is a key regulatory enzyme in the coagulation cascade; itserves a pluralistic role as both a positive and negative feedbackregulator. In addition to its direct effect on hemostasis, thrombinexerts direct effects on diverse cell types that support and amplifypathogenesis of arterial thrombus disease. The enzyme is the strongestactivator of platelets causing them to aggregate and release substances(eg. ADP TXA.sub.2 NE) that further propagate the thrombotic cycle.Platelets in a fibrin mesh comprise the principal framework of a whitethrombus. Thrombin also exerts direct effects on endothelial cellscausing release of vasoconstrictor substances and translocation ofadhesion molecules that become sites for attachment of immune cells. Inaddition, the enzyme causes mitogenesis of smooth muscle cells andproliferation of fibroblasts. From this analysis, it is apparent thatinhibition of thrombin activity by thrombin inhibitors constitutes aviable therapeutic approach towards the attenuation of proliferativeevents associated with thrombosis.

Toxins (SEQ ID NOS: 1378-1415)

A toxin can be conjugated using the present invention to target cancercells, receptors, viruses, or blood cells. Once the toxin binds to thetarget cells the toxin is allowed to internalize and cause cell toxicityand eventually cell death. Toxins have been widely used as cancertherapeutics.

One example of a class of toxins is the mast cell degranulating peptide,a cationic 22-amino acid residue peptide with two disulfide bridgesisolated from bee venom, causes mast cell degranulation and histaminerelease at low concentrations and has anti-inflammatory activity athigher concentrations. It is a powerful anti-inflammatory, more than 100times more effective than hydrocortisone in reducing inflammation.Because of these unique immunologic properties, MCD peptide may serve asa useful tool for studying secretory mechanisms of inflammatory cellssuch as mast cells, basophils, and leukocytes, leading to the design ofcompounds with therapeutic potential. An example of a mast celldegranulating peptide is mastoparans, originating from wasp venom. Itdegranulates mast cells in the concentration of 0.5 μg/ml and releaseshistamine from the cells in the same concentration. See I Y. Hirai etal., Chem. Pharm. Bull. 27, 1942 (1979).

Other examples of such toxins include omega-agatoxin TK, agelenin,apamin, calcicudine, calciseptine, charbdotoxin, chlorotoxin,conotoxins, endotoxin inhibitors, gegraphutoxins, iberiotoxin,kaliotoxin, mast cell degranulating peptides, margatoxin, neurotoxinNSTX-3, PLTX-II, scyllatoxin, stichodactyla toxin, and derivatives andfragments thereof.

Trypsin Inhibitors (SEQ ID NOS: 1416-1418)

Trypsin inhibitors functions as an inhibitors of trypsin, as well asother serine proteases. Useful for treatment of lung inflammation,pancreatitis, myocardial infarction, cerebrovascular ischemia

Virus Related Peptides (SEQ ID NOS: 1419-1529)

Virus related peptides are proteins related to viruses, for examplevirus receptors, virus inhibitors, and envelope proteins. Examplesinclude but are not limited to peptide inhibitors of humanimmunodeficiency virus (HIV), respiratory syncytial virus (RSV), humanparainfluenza virus (HPV), measles virus (MeV), and simianimmunodeficiency virus (SIV), fluorogenic Human CMV Protease Substrate,HCV Core Protein, HCV NS4A Protein, Hepatitis B Virus Receptor BindingFragment, Hepatitis B Virus Pre-S Region, Herpes Virus Inhibitor 2, HIVEnvelope Protein Fragment, HIV gag fragment, HIV substrate, HIV-1Inhibitory Peptide, peptide T, T21, V3 decapeptide, Virus ReplicationInhibitor Peptide, and their fragments and derivatives.

These peptides can be administered therapeutically. For example, peptideT is a chain of 8 amino acids from the V2 region of HIV-1 gp120. Theseamino acids look like a portion of HIV's outer envelope. It is underinvestigation as a treatment for HIV-related neurological andconstitutional symptoms, as peptide T may be able to alleviate symptomslike fevers, night sweats, weight loss, and fatigue. It has also beenshown to resolve psoriatic lesions.

Miscellaneous Peptides (SEQ ID NOS: 1529-1617)

Including adjuvant peptide analogs, alpha mating factor, antiarrhythmicpeptide, anorexigenic peptide, alpha-1 antitrypsin, bovine pinealantireproductive peptide, bursin, C3 peptide P16, cadherin peptide,chromogranin A fragment, contraceptive tetrapeptide, conantokin G,conantokin T, crustacean cardioactive peptide, C-telopeptide, cytochromeb588 peptide, decorsin, delicious peptide, delta-sleep-inducing peptide,diazempam-binding inhibitor fragment, nitric oxide synthase blockingpeptide, OVA peptide, platelet calpain inhibitor (P1), plasminogenactivator inhibitor 1, rigin, schizophrenia related peptide, sodiumpotassium Atherapeutic peptidease inhibitor-1, speract, sperm activatingpeptide, systemin, thrombin receptor agonist (three peptides), tuftsin,adipokinetic hormone, uremic pentapeptide, Antifreeze Polypeptide, tumornecrosis factor, leech [Des Asp10]Decorsin, L-OrnithyltaurineHydrochloride, p-Aminophenylacetyl Tuftsin,Ac-Glu-Glu-Val-Val-Ala-Cys-pNA, Ac-Ser-Asp-Lys-Pro, Ac-rfwink-NH2,Cys-Gly-Tyr-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Gly-Gly, DAla-Leu,D-D-D-D-D, D-D-D-D-D-D, N-P-N-A-N-P-N-A, V-A-I-T-V-L-V-K, V-G-V-R-V-R,V-I-H-S, V-P-D-P-R, Val-Thr-Cys-Gly, R-S-R, Sea Urchin Sperm ActivatingPeptide, SHU-9119 MC3-R & MC4-R Antagonist, glaspimod (immunostimulant,useful against bacterial infections, fungal infections, immunedeficiency immune disorder, leukopenia), HP-228 (melanocortin, usefulagainst chemotherapy induced emesis, toxicity, pain, diabetes mellitus,inflammation, rheumatoid arthritis, obesity), alpha 2-plasmin inhibitor(plasmin inhibitor), APC tumor suppressor (tumor suppressor, usefulagainst neoplasm), early pregnancy factor (immunosuppressor), endozepinediazepam binding inhibitor (receptor peptide), gamma interferon (usefulagainst leukemia), glandular kallikrei n-1 (immunostimulant), placentalribonuclease inhibitor, sarcolecin binding protein, surfactant proteinD, wilms' tumor suppressor, wilm's tumor suppressor, GABAB 1b receptorpeptide, prion related peptide (iPrP13), choline binding proteinfragment (bacterial related peptide), telomerase inhibitor, cardiostatinpeptide, endostatin derived peptide (angiogenesis inhibitor), prioninhibiting peptide, N-methyl D-aspartate receptor antagonist, C-peptideanalog (useful against diabetic complications).

2. Modified Therapeutic Peptides

This invention relates to modified therapeutic peptides and theirderivatives. The modified therapeutic peptides of the invention includereactive groups which can react with available reactive functionalitieson blood components to form covalent bonds. The invention also relatesto such modifications, such combinations with blood components andmethods for their use. These methods include extending the effectivetherapeutic in vivo half life of the modified therapeutic peptides.

To form covalent bonds with functionalities on a protein, one may use asa reactive group a wide variety of active carboxyl groups, particularlyesters, where the hydroxyl moiety is physiologically acceptable at thelevels required to modify the therapeutic peptide. While a number ofdifferent hydroxyl groups may be employed in these linking agents, themost convenient would be N-hydroxysuccinimide (NHS), andN-hydroxy-sulfosuccinimide (sulfo-NHS).

Primary amines are the principal targets for NHS esters as diagramed inschematic 1A below. Accessible α-amine groups present on the N-terminiof proteins react with NHS esters. However, α-amino groups on a proteinmay not be desirable or available for the NHS coupling. While five aminoacids have nitrogen in their side chains, only the ε-amine of lysinereacts significantly with NHS esters. An amide bond is formed when theNHS ester conjugation reaction reacts with primary amines releasingN-hydroxysuccinimide as demonstrated in schematic 1A below.

In the preferred embodiments of this invention, the functionality on theprotein will be a thiol group and the reactive group will be amaleimido-containing group such as gamma-maleimide-butyralamide (GMBA)or MPA. The maleimido group is most selective for sulfhydryl groups onpeptides when the pH of the reaction mixture is kept between 6.5 and 7.4as shown in schematic 1B below. At pH 7.0, the rate of reaction ofmaleimido groups with sulfhydryls is 1000-fold faster than with amines.A stable thioether linkage between the maleimido group and thesulfhydryl is formed which cannot be cleaved under physiologicalconditions.

The therapeutic peptides and peptide derivatives of the invention may bemodified for specific labeling and non-specific labeling of bloodcomponents.

A. Specific Labeling

Preferably, the therapeutic peptides of this invention are designed tospecifically react with thiol groups on mobile blood proteins. Suchreaction is preferably established by covalent bonding of a therapeuticpeptide modified with a maleimide link (e.g. prepared from GMBS, MPA orother maleimides) to a thiol group on a mobile blood protein such asserum albumin or IgG.

Under certain circumstances, specific labeling with maleimides offersseveral advantages over non-specific labeling of mobile proteins withgroups such as NHS and sulfo-NHS. Thiol groups are less abundant in vivothan amino groups. Therefore, the maleimide derivatives of thisinvention will covalently bond to fewer proteins. For example, inalbumin (the most abundant blood protein) there is only a single thiolgroup. Thus, therapeutic peptide-maleimide-albumin conjugates will tendto comprise approximately a 1:1 molar ratio of therapeutic peptide toalbumin. In addition to albumin, IgG molecules (class II) also have freethiols. Since IgG molecules and serum albumin make up the majority ofthe soluble protein in blood they also make up the majority of the freethiol groups in blood that are available to covalently bond tomaleimide-modified therapeutic peptides.

Further, even among free thiol-containing blood proteins, specificlabeling with maleimides leads to the preferential formation oftherapeutic peptide-maleimide-albumin conjugates, due to the uniquecharacteristics of albumin itself. The single free thiol group ofalbumin, highly conserved among species, is located at amino acidresidue 34 (Cys³⁴). It has been demonstrated recently that the Cys³⁴ ofalbumin has increased reactivity relative to free thiols on other freethiol-containing proteins. This is due in part to the very low pK valueof 5.5 for the Cys³⁴ of albumin. This is much lower than typical pKvalues for cysteines residues in general, which are typically about 8.Due to this low pK, under normal physiological conditions Cys³⁴ ofalbumin is predominantly in the ionized form, which dramaticallyincreases its reactivity. In addition to the low pK value of Cys³⁴,another factor which enhances the reactivity of Cys³⁴ is its location,which is in a crevice close to the surface of one loop of region V ofalbumin. This location makes Cys³⁴ very available to ligands of allkinds, and is an important factor in Cys³⁴'s biological role as freeradical trap and free thiol scavenger. These properties make Cys³⁴highly reactive with therapeutic peptide-maleimides, and the reactionrate acceleration can be as much as 1000-fold relative to rates ofreaction of therapeutic peptide-maleimides with other free-thiolcontaining proteins.

Another advantage of therapeutic peptide-maleimide-albumin conjugates isthe reproducibility associated with the 1:1 loading of peptide toalbumin specifically at Cys³⁴. Other techniques, such as glutaraldehyde,DCC, EDC and other chemical activations of, for example, free amineslack this selectivity. For example, albumin contains 52 lysine residues,25-30 of which are located on the surface of albumin and accessible forconjugation. Activating these lysine residues, or alternativelymodifying peptides to couple through these lysine residues, results in aheterogenous population of conjugates. Even if 1:1 molar ratios ofpeptide to albumin are employed, the yield will consist of multipleconjugation products, some containing 0, 1, 2 or more peptides peralbumin, and each having peptides randomly coupled at any one of the25-30 available lysine sites. Given the numerous combinations possible,characterization of the exact composition and nature of each batchbecomes difficult, and batch-to-batch reproducibility is all butimpossible, making such conjugates less desirable as a therapeutic.Additionally, while it would seem that conjugation through lysineresidues of albumin would at least have the advantage of delivering moretherapeutic agent per albumin molecule, studies have shown that a 1:1ratio of therapeutic agent to albumin is preferred. In an article byStehle, et al., “The Loading Rate Determines Tumor Targeting Propertiesof Methotrexate-Albumin Conjugates in Rats,” Anti-Cancer Drugs, Vol. 8,pp. 677-685 (1997), incorporated herein in its entirety, the authorsreport that a 1:1 ratio of the anti-cancer methotrexate to albuminconjugated via glutaraldehyde gave the most promising results. Theseconjugates were taken up by tumor cells, whereas conjugates bearing 5:1to 20:1 methotrexate molecules had altered HPLC profiles and werequickly taken up by the liver in vivo. It is postulated that at thesehigher ratios, conformational changes to albumin diminish itseffectiveness as a therapeutic carrier.

Through controlled administration of maleimide-therapeutic peptides invivo, one can control the specific labeling of albumin and IgG in vivo.In typical administrations, 80-90% of the administeredmaleimide-therapeutic peptides will label albumin and less than 5% willlabel IgG. Trace labeling of free thiols such as glutathione will alsooccur. Such specific labeling is preferred for in vivo use as it permitsan accurate calculation of the estimated half-life of the administeredagent.

In addition to providing controlled specific in vivo labeling,maleimide-therapeutic peptides can provide specific labeling of serumalbumin and IgG ex vivo. Such ex vivo labeling involves the addition ofmaleimide-therapeutic peptides to blood, serum or saline solutioncontaining serum albumin and/or IgG. Once modified ex vivo withmaleimide-therapeutic peptides, the blood, serum or saline solution canbe readministered to the blood for in vivo treatment.

In contrast to NHS-peptides, maleimide-therapeutic peptides aregenerally quite stable in the presence of aqueous solutions and in thepresence of free amines. Since maleimide-therapeutic peptides will onlyreact with free thiols, protective groups are generally not necessary toprevent the maleimide-therapeutic peptides from reacting with itself. Inaddition, the increased stability of the peptide permits the use offurther purification steps such as HPLC to prepare highly purifiedproducts suitable for in vivo use. Lastly, the increased chemicalstability provides a product with a longer shelf life.

B. Nonspecific Labeling

The therapeutic peptides of the invention may also be modified fornon-specific labeling of blood components. Bonds to amino groups willgenerally be employed, particularly with the formation of amide bondsfor non-specific labeling. To form such bonds, one may use as achemically reactive group coupled to the therapeutic peptide a widevariety of active carboxyl groups, particularly esters, where thehydroxyl moiety is physiologically acceptable at the levels required.While a number of different hydroxyl groups may be employed in theselinking agents, the most convenient would be N-hydroxysuccinimide (NHS)and N-hydroxy-sulfosuccinimide (sulfo-NHS).

Other linking agents which may be utilized are described in U.S. Pat.No. 5,612,034, which is hereby incorporated herein.

The various sites with which the chemically reactive groups of thenon-specific therapeutic peptides may react in vivo include cells,particularly red blood cells (erythrocytes) and platelets, and proteins,such as immunoglobulins, including IgG and IgM, serum albumin, ferritin,steroid binding proteins, transferrin, thyroxin binding protein,α-2-macroglobulin, and the like. Those receptors with which thederivatized therapeutic peptides react, which are not long-lived, willgenerally be eliminated from the human host within about three days. Theproteins indicated above (including the proteins of the cells) willremain in the bloodstream at least three days, and may remain five daysor more (usually not exceeding 60 days, more usually not exceeding 30days) particularly as to the half life, based on the concentration inthe blood.

For the most part, reaction will be with mobile components in the blood,particularly blood proteins and cells, more particularly blood proteinsand erythrocytes. By “mobile” is intended that the component does nothave a fixed situs for any extended period of time, generally notexceeding 5 minutes, more usually one minute, although some of the bloodcomponents may be relatively stationary for extended periods of time.Initially, there will be a relatively heterogeneous population oflabeled proteins and cells. However, for the most part, the populationwithin a few days after administration will vary substantially from theinitial population, depending upon the half-life of the labeled proteinsin the blood stream. Therefore, usually within about three days or more,IgG will become the predominant labeled protein in the blood stream.

Usually, by day 5 post-administration, IgG, serum albumin anderythrocytes will be at least about 60 mole %, usually at least about 75mole %, of the conjugated components in blood, with IgG, IgM (to asubstantially lesser extent) and serum albumin being at least about 50mole %, usually at least about 75 mole %, more usually at least about 80mole %, of the non-cellular conjugated components.

The desired conjugates of non-specific therapeutic peptides to bloodcomponents may be prepared in vivo by administration of the therapeuticpeptides directly to the patient, which may be a human or other mammal.The administration may be done in the form of a bolus or introducedslowly over time by infusion using metered flow or the like.

If desired, the subject conjugates may also be prepared ex vivo bycombining blood with modified therapeutic peptides of the presentinvention, allowing covalent bonding of the modified therapeuticpeptides to reactive functionalities on blood components and thenreturning or administering the conjugated blood to the host. Moreover,the above may also be accomplished by first purifying an individualblood component or limited number of components, such as red bloodcells, immunoglobulins, serum albumin, or the like, and combining thecomponent or components ex vivo with the chemically reactive therapeuticpeptides. The labeled blood or blood component may then be returned tothe host to provide in vivo the subject therapeutically effectiveconjugates. The blood also may be treated to prevent coagulation duringhandling ex vivo.

3. Synthesis of Therapeutic Peptides Used in the Present Invention

Peptide fragments may be synthesized by standard methods of solid phasepeptide chemistry known to those of ordinary skill in the art. Forexample, peptide fragments may be synthesized by solid phase chemistrytechniques following the procedures described by Steward and Young(Steward, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2ndEd., Pierce Chemical Company, Rockford, Ill., (1984) using an AppliedBiosystem synthesizer. Similarly, multiple fragments may be synthesizedthen linked together to form larger fragments. These synthetic peptidefragments can also be made with amino acid substitutions at specificlocations.

For solid phase peptide synthesis, a summary of the many techniques maybe found in J. M. Stewart and J. D. Young, Solid Phase PeptideSynthesis, W. H. Freeman Co. (San Francisco), 1963 and J. Meienhofer,Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (NewYork), 1973. For classical solution synthesis see G. Schroder and K.Lupke, The Peptides, Vol. 1, Acacemic Press (New York). In general,these methods comprise the sequential addition of one or more aminoacids or suitably protected amino acids to a growing peptide chain.Normally, either the amino or carboxyl group of the first amino acid isprotected by a suitable protecting group. The protected or derivatizedamino acid is then either attached to an inert solid support or utilizedin solution by adding the next amino acid in the sequence having thecomplimentary (amino or carboxyl) group suitably protected and underconditions suitable for forming the amide linkage. The protecting groupis then removed from this newly added amino acid residue and the nextamino acid (suitably protected) is added, and so forth.

After all the desired amino acids have been linked in the propersequence, any remaining protecting groups (and any solid support) areremoved sequentially or concurrently to afford the final polypeptide. Bysimple modification of this general procedure, it is possible to addmore than one amino acid at a time to a growing chain, for example, bycoupling (under conditions which do not racemize chiral centers) aprotected tripeptide with a properly protected dipeptide to form, afterdeprotection, a pentapeptide.

A particularly preferred method of preparing compounds of the presentinvention involves solid phase peptide synthesis wherein the amino acidα-N-terminal is protected by an acid or base sensitive group. Suchprotecting groups should have the properties of being stable to theconditions of peptide linkage formation while being readily removablewithout destruction of the growing peptide chain or racemization of anyof the chiral centers contained therein. Suitable protecting groups are9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc),benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl,t-amyloxycarbonyl, isobornyloxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl,2-cyano-t-butyloxycarbonyl, and the like. The9-fluorenyl-methyloxycarbonyl (Fmoc) protecting group is particularlypreferred for the synthesis of Itherapeutic peptide fragments. Otherpreferred side chain protecting groups are, for side chain amino groupslike lysine and arginine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc),nitro, p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc, andadamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxycarbonyl,2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyland acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; forhistidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl;for tryptophan, formyl; for asparticacid and glutamic acid, benzyl andt-butyl and for cysteine, triphenylmethyl (trityl).

In the solid phase peptide synthesis method, the α-C-terminal amino acidis attached to a suitable solid support or resin. Suitable solidsupports useful for the above synthesis are those materials which areinert to the reagents and reaction conditions of the stepwisecondensation-deprotection reactions, as well as being insoluble in themedia used. The preferred solid support for synthesis of α-C-terminalcarboxy peptides is 4-hydroxymethylphenoxymethyl-copoly(styrene-1%divinylbenzene). The preferred solid support for α-C-terminal amidepeptides is the4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resinavailable from Applied Biosystems (Foster City, Calif.). Theα-C-terminal amino acid is coupled to the resin by means ofN,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC)or O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium-hexafluorophosphate(HBTU), with or without 4-dimethylaminopyridine (DMAP),1-hydroxybenzotriazole (HOBT),benzotriazol-1-yloxy-tris(dimethylamino)phosphonium-hexafluorophosphate(BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCI), mediatedcoupling for from about 1 to about 24 hours at a temperature of between10° and 50° C. in a solvent such as dichloromethane or DMF.

When the solid support is4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin,the Fmoc group is cleaved with a secondary amine, preferably piperidine,prior to coupling with the α-C-terminal amino acid as described above.The preferred method for coupling to the deprotected4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resinis O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluoro-phosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. Thecoupling of successive protected amino acids can be carried out in anautomatic polypeptide synthesizer as is well known in the art. In apreferred embodiment, the α-N-terminal amino acids of the growingpeptide chain are protected with Fmoc. The removal of the Fmocprotecting group from the α-N-terminal side of the growing peptide isaccomplished by treatment with a secondary amine, preferably piperidine.Each protected amino acid is then introduced in about 3-fold molarexcess, and the coupling is preferably carried out in DMF. The couplingagent is normallyO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT,1 equiv.).

At the end of the solid phase synthesis, the polypeptide is removed fromthe resin and deprotected, either in successively or in a singleoperation. Removal of the polypeptide and deprotection can beaccomplished in a single operation by treating the resin-boundpolypeptide with a cleavage reagent comprising thianisole, water,ethanedithiol and trifluoroacetic acid. In cases wherein theα-C-terminal of the polypeptide is an alkylamide, the resin is cleavedby aminolysis with an alkylamine. Alternatively, the peptide may beremoved by transesterification, e.g. with methanol, followed byaminolysis or by direct transamidation. The protected peptide may bepurified at this point or taken to the next step directly. The removalof the side chain protecting groups is accomplished using the cleavagecocktail described above. The fully deprotected peptide is purified by asequence of chromatographic steps employing any or all of the followingtypes: ion exchange on a weakly basic resin (acetate form); hydrophobicadsorption chromatography on underivitized polystyrene-divinylbenzene(for example, Amberlite XAD); silica gel adsorption chromatography; ionexchange chromatography on carboxymethylcellulose; partitionchromatography, e.g. on Sephadex G-25, LH-20 or countercurrentdistribution; high performance liquid chromatography (HPLC), especiallyreverse-phase HPLC on octyl- or octadecylsilyl-silica bonded phasecolumn packing.

Molecular weights of these therapeutic peptides are determined usingFast Atom Bombardment (FAB) Mass Spectroscopy.

The therapeutic peptides of the invention may be synthesized with N- andC-terminal protecting groups for use as pro-drugs.

(1) N-Terminal Protective Groups

As discussed above, the term “N-protecting group” refers to those groupsintended to protect the α-N-terminal of an amino acid or peptide or tootherwise protect the amino group of an amino acid or peptide againstundesirable reactions during synthetic procedures. Commonly usedN-protecting groups are disclosed in Greene, “Protective Groups InOrganic Synthesis,” (John Wiley & Sons, New York (1981)), which ishereby incorporated by reference. Additionally, protecting groups can beused as pro-drugs which are readily cleaved in vivo, for example, byenzymatic hydrolysis, to release the biologically active parent.α-N-protecting groups comprise loweralkanoyl groups such as formyl,acetyl (“Ac”), propionyl, pivaloyl, t-butylacetyl and the like; otheracyl groups include 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl,trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, -chlorobutyryl,benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and the like;sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-ethoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and thelike; arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl,9-fluorenylmethyloxycarbonyl (Fmoc) and the like and silyl groups suchas trimethylsilyl and the like.

(2) Carboxy Protective Groups

As discussed above, the term “carboxy protecting group” refers to acarboxylic acid protecting ester or amide group employed to block orprotect the carboxylic acid functionality while the reactions involvingother functional sites of the compound are performed. Carboxy protectinggroups are disclosed in Greene, “Protective Groups in Organic Synthesis”pp. 152-186 (1981), which is hereby incorporated by reference.Additionally, a carboxy protecting group can be used as a pro-drugwhereby the carboxy protecting group can be readily cleaved in vivo, forexample by enzymatic hydrolysis, to release the biologically activeparent. Such carboxy protecting groups are well known to those skilledin the art, having been extensively used in the protection of carboxylgroups in the penicillin and cephalosporin fields as described in U.S.Pat. Nos. 3,840,556 and 3,719,667, the disclosures of which are herebyincorporated herein by reference. Representative carboxy protectinggroups are C₁-C₈ loweralkyl (e.g., methyl, ethyl or t-butyl and thelike); arylalkyl such as phenethyl or benzyl and substituted derivativesthereof such as alkoxybenzyl or nitrobenzyl groups and the like;arylalkenyl such as phenylethenyl and the like; aryl and substitutedderivatives thereofsuch as 5-indanyl and the like; dialkylaminoalkylsuch as dimethylaminoethyl and the like); alkanoyloxyalkyl groups suchas acetoxymethyl, butyryloxymethyl, valeryloxymethyl,isobutyryloxymethyl, isovaleryloxymethyl, 1-(propionyloxy)-1-ethyl,1-(pivaloyloxyl)-1-ethyl, 1-methyl-1-(propionyloxy)-1-ethyl,pivaloyloxymethyl, propionyloxymethyl and the like;cycloalkanoyloxyalkyl groups such as cyclopropylcarbonyloxymethyl,cyclobutylcarbonyloxymethyl, cyclopentylcarbonyloxymethyl,cyclohexylcarbonyloxymethyl and the like; aroyloxyalkyl such asbenzoyloxymethyl, benzoyloxyethyl and the like;arylalkylcarbonyloxyalkyl such as benzylcarbonyloxymethyl,2-benzylcarbonyloxyethyl and the like; alkoxycarbonylalkyl orcycloalkyloxycarbonylalkyl such as methoxycarbonylmethyl,cyclohexyloxycarbonylmethyl, 1-methoxycarbonyl-1-ethyl and the like;alkoxycarbonyloxyalkyl or cycloalkyloxycarbonyloxyalkyl such asmethoxycarbonyloxymethyl, t-butyloxycarbonyloxymethyl,1-ethoxycarbonyloxy-1-ethyl, 1-cyclohexyloxycarbonyloxy-1-ethyl and thelike; aryloxycarbonyloxyalkyl such as 2-(phenoxycarbonyloxy)ethyl,2-(5-indanyloxycarbonyloxy)ethyl and the like;alkoxyalkylcarbonyloxyalkyl such as2-(1-methoxy-2-methylpropan-2-oyloxy)ethyl and like;arylalkyloxycarbonyloxyalkyl such as 2-(benzyloxycarbonyloxy)ethyl andthe like; arylalkenyloxycarbonyloxyalkyl such as2-(3-phenylpropen-2-yloxycarbonyloxy)ethyl and the like;alkoxycarbornylaminoalkyl such as t-butyloxycarbonylaminomethyl and thelike; alkylaminocarbonylaminoalkyl such asmethylaminocarbonylaminomethyl and the like; alkanoylaminoalkyl such asacetylaminomethyl and the like; heterocycliccarbonyloxyalkyl such as4-methylpiperazinylcarbonyloxymethyl and the like;dialkylaminocarbonylalkyl such as dimethylaminocarbonylmethyl,diethylaminocarbonylmethyl and the like;(5-(loweralkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl such as(5-t-butyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like; and(5-phenyl-2-oxo-1,3-dioxolen-4-yl)alkyl such as(5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like.

Representative amide carboxy protecting groups are aminocarbonyl andloweralkylaminocarbonyl groups.

Preferred carboxy-protected compounds of the invention are compoundswherein the protected carboxy group is a loweralkyl, cycloalkyl orarylalkyl ester, for example, methyl ester, ethyl ester, propyl ester,isopropyl ester, butyl ester, sec-butyl ester, isobutyl ester, amylester, isoamyl ester, octyl ester, cyclohexyl ester, phenylethyl esterand the like or an alkanoyloxyalkyl, cycloalkanoyloxyalkyl,aroyloxyalkyl or an arylalkylcarbonyloxyalkyl ester. Preferred amidecarboxy protecting groups are loweralkylaminocarbonyl groups. Forexample, aspartic acid may be protected at the α-C-terminal by an acidlabile group (e.g. t-butyl) and protected at the β-C-terminal by ahydrogenation labile group (e.g. benzyl) then deprotected selectivelyduring synthesis.

Alternatively, it is also possible to obtain fragments of the peptidesby fragmenting the naturally occurring amino acid sequence, using, forexample, a proteolytic enzyme according to methods well known in theart. Further, it is possible to obtain the desired fragments of thetherapeutic peptide through the use of recombinant DNA technology usingmethods well known in the art.

4. Modification of Therapeutic Peptides

The manner of producing the modified therapeutic peptides of the presentinvention will vary widely, depending upon the nature of the variouselements comprising the molecule. The synthetic procedures will beselected so as to be simple, provide for high yields, and allow for ahighly purified stable product. Normally, the reactive group will becreated as the last stage, for example, with a carboxyl group,esterification to form an active ester will be the last step of thesynthesis. Specific methods for the production of modified therapeuticpeptides of the present invention are described below.

Generally, the modified therapeutic peptides of the present inventionmay be made using blind or structure activity relationship (SAR) drivensubstitution. SAR is an analysis which defines the relationship betweenthe structure of a molecule and its pharmacological activity for aseries of compounds. Various studies relative to individual therapeuticpeptides show how the activity of the peptide varies according to thevariation of chemical structure or chemical properties. Morespecifically, first the therapeutic activity of the free peptide isassayed. Next, the peptide is modified according to the invention,either at the N-terminus, at the C-terminus, or in the interior of thepeptide with the linking group only. The linking group will include thereactive group as discussed above. The therapeutic activity of thismodified peptide-linking group is assayed next, and based on thedetected activity a decision is made regarding the modification site.Next, the peptide conjugate is prepared and its therapeutic ativity isdetermined. If the therapeutic activity of the peptide after conjugationis not substantially reduced (i.e. if the therapeutic activity isreduced by less than 10 fold), then the stability of the peptide ismeasured as indicated by its in vivo lifetime. If the stability is notimproved to a desired level, then the peptide is modified at analternative site, and the procedure is repeated until a desired level oftherapeutic activity and a desired stability are achieved.

More specifically, each therapeutic peptide selected to undergo thederivatization with a linker and a reactive group will be modifiedaccording to the following criteria: if a terminal carboxylic group isavailable on the therapeutic peptide and is not critical for theretention of pharmacological activity, and no other sensitive functionalgroup is present on the therapeutic peptide, then the carboxylic acidwill be chosen as attachment point for the linker-reactive groupmodification. If the terminal carboxylic group is involved inpharmacological activity, or if no carboxylic acids are available, thenany other sensitive functional group not critical for the retention ofpharmacological activity will be selected as the attachment point forthe linker-reactive group modification. If several sensitive functionalgroups are available on a therapeutic peptide, a combination ofprotecting groups will be used in such a way that after addition of thelinker/reactive group and deprotection of all the protected sensitivefunctional groups, retention of pharmacological activity is stillobtained. If no sensitive functional groups are available on thetherapeutic peptide, or if a simpler modification route is desired,synthetic efforts will allow for a modification of the original peptidein such a way that retention of biological activity and retention ofreceptor or target specificity is obtained. In this case themodification will occur at the opposite end of the peptide.

An NHS derivative may be synthesized from a carboxylic acid in absenceof other sensitive functional groups in the therapeutic peptide.Specifically, such a therapeutic peptide is reacted withN-hydroxysuccinimide in anhydrous CH₂Cl₂ and EDC, and the product ispurified by chromatography or recrystallized from the appropriatesolvent system to give the NHS derivative.

Alternatively, an NHS derivative may be synthesized from a therapeuticpeptide that contains an amino and/or thiol group and a carboxylic acid.When a free amino or thiol group is present in the molecule, it ispreferable to protect these sensitive functional groups prior to performthe addition of the NHS derivative. For instance, if the moleculecontains a free amino group, a transformation of the amine into a Fmocor preferably into a tBoc protected amine is necessary prior to performthe chemistry described above. The amine functionality will not bedeprotected after preparation of the NHS derivative. Therefore thismethod applies only to a compound whose amine group is not required tobe freed to induce a pharmacological desired effect. If the amino groupneeds to be freed to retain the original biological properties of themolecule, then another type of chemistry described in example 3-6 has tobe performed.

In addition, an NHS derivative may be synthesized from a therapeuticpeptide containing an amino or a thiol group and no carboxylic acid.When the selected molecule contains no carboxylic acid, an array ofbifunctional linkers can be used to convert the molecule into a reactiveNHS derivative. For instance, ethylene glycol-bis(succinimydylsuccinate)(EGS) and triethylamine dissolved in DMF and added to the free aminocontaining molecule (with a ratio of 10:1 in favor of EGS) will producethe mono NHS derivative. To produce an NHS derivative from a thiolderivatized molecule, one can use N-[-maleimidobutyryloxy]succinimideester (GMBS) and triethylamine in DMF. The maleimido group will reactwith the free thiol and the NHS derivative will be purified from thereaction mixture by chromatography on silica or by HPLC.

An NHS derivative may also be synthesized from a therapeutic peptidecontaining multiple sensitive functional groups. Each case will have tobe analyzed and solved in a different manner. However, thanks to thelarge array of protecting groups and bifunctional linkers that arecommercially available, this invention is applicable to any therapeuticpeptide with preferably one chemical step only to derivatize thetherapeutic peptide (as described in example 1 or 3) or two steps (asdescribed in example 2 and involving prior protection of a sensitivegroup) or three steps (protection, activation and deprotection). Underexceptional circumstances only, would we require to use multiple steps(beyond three steps) synthesis to transform a therapeutic peptide intoan active NHS or maleimide derivative.

A maleimide derivative may also be synthesized from a therapeuticpeptide containing a free amino group and a free carboxylic acid. Toproduce a maleimide derivative from a amino derivatized molecule, onecan use N-[γ-maleimidobutyryloxy]succinimide ester (GMBS) andtriethylamine in DMF. The succinimide ester group will react with thefree amino and the maleimide derivative will be purified from thereaction mixture by crystallization or by chromatography on silica or byHPLC.

Finally, a maleimide derivative may be synthesized from a therapeuticpeptide containing multiple other sensitive functional groups and nofree carboxylic acids. When the selected molecule contains no carboxylicacid, an array of bifunctional crosslinking reagents can be used toconvert the molecule into a reactive NHS derivative. For instancemaleimidopropionic acid (MPA) can be coupled to the free amine toproduce a maleimide derivative through reaction of the free amine withthe carboxylic group of MPA using HBTU/HOBt/DIEA activation in DMF.Alternatively, a lysine residue can be added on the C-terminus end ofthe peptide to allow for conjugation onto the -amino group of the lysineas described in the examples below. This added lysine allows for simpleand efficient modification at the C-terminus of the peptide whilekeeping the terminal end capped by an amide function as designed by theinitial choice of the resin

Many other commercially available heterobifunctional crosslinkingreagents can alternatively be used when needed. A large number ofbifunctional compounds are available for linking to entities.Illustrative reagents include: azidobenzoyl hydrazide,N-[4-(p-azidosalicylamino)butyl]-3′-[2′-pyridyidithio)propionamide),bis-sulfosuccinimidyl suberate, dimethyl adipimidate, disuccinimidyltartrate, N-y-maleimidobutyryloxysuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate,N-succinimidyl[4-azidophenyl]-1,3′-dithiopropionate,N-succinimidyl[4-iodoacetyl]aminobenzoate, glutaraldehyde, andsuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate.

Even more specifically, the peptides are preferably modified accordingto the nature of their substituants and the presence or absence of freecysteines. Most peptides can be gathered into three distinct categories:(1) peptides that contain no cysteines; (2) peptides that contain onecysteine, (3) peptides that contain two cysteines as a disulfide bridge(cystine); and (4) peptides that contain multiple cysteines.

A. Peptides that Contain No Cysteines

Where the peptide contains no cysteine, addition from the C terminus isperformed with all residues cleaved from the support resin and fullyprotected. Solution phase activation of C-terminus with EDC and NHS canbe reacted with an amino-alkyl-maleimide in one pot. The peptide is thenfully deprotected. Alternatively, a lysine residue can be added on theC-terminus of the peptide to allow modification at the epsilon aminogroup of the lysine while keeping the carboxy terminus capped with anamide group. Such an addition of a lysine residue is preferablyperformed only where the addition does not substantially affect thetherapeutic activity of the peptide. The generalized reaction scheme forC-terminus modification of peptides that contain no cysteinesillustrated in the schematic diagram below.

If an N-terminus modification is favored, and again for a peptidecontaining no cysteine, addition on the N terminus is performed with allresidues still on the support resin and fully protected. Addition ofactivated NHS-Mal bifunctional linker could be performed on deprotectedN-terminus with peptide still on resin. The peptide is then fullydeprotected. Examples of therapeutic peptides that contain no cysteinand undergo a C-terminus modification are described in examples 7-26.Examples of therapeutic peptides that contain no cystein and undergo aN-terminus modification are described in examples 27-38. The generalizedreaction scheme for N-terminus modification of peptides that contain nocysteines is illustrated in the schematic diagrams below, using heteroNHS maleimide (GMBS like) and 3-MPA, respectively.

Alternatively, the peptide may be modified at an internal amino acid(i.e. neither at the C-terminus nor at the N-terminus). The generalizedreaction scheme for modification at an internal amino acid of a peptidethat contains no free cysteines is illustrated in the schematic diagramsbelow, using homo bis NHS and hetero NHS maleimide.

Peptides that contain no cysteine and can be modified as described aboveinclude fragments of the Kringle 5 peptide, of the GLP-1 peptide, ofdynorphin A, human growth hormone releasing factor, the 1-24 fragment ofhuman neuropeptide Y, and human secretin. Full description of thechemistry for each of these peptides is reported in the Example section.

B. Peptides that Contain One Cysteine

Where the peptide contains one cysteine, the cysteine must stay cappedafter addition of the maleimide. If the cysteine is involved in bindingsite, assessment has to be made of how much potency is lost is cysteineis capped by a protecting group. If the cysteine can stay capped, thenthe synthetic path is similar to that described in section A above foreither a C or an N terminus modification.

Alternatively, the peptide may be modified at an internal amino acid(i.e. neither at the C-terminus nor at the N-terminus). The generalizedreaction scheme for modification at an internal amino acid of a peptidethat contains no cysteines is illustrated in the schematic diagrambelow, using homobis maleimide and hetero NHS maleimide (GMBS like).

Examples of therapeutic peptides that contain one cysteine include G_(α)(the alpha subunit of Gtherapeutic peptide binding protein), the 724-739fragment of rat brain nitric oxide synthase blocking peptide, the alphasubunit 1-32 fragment of human [Tyr0] inhibin, the 254-274 fragment ofHIV envelope protein, and P34cdc2 kinase fragment.

C. Peptides that Contain Two Cysteines as a Disulfide Bridge (Cystine)

Where the peptide contains two cysteines as a disulfide bridge, thepeptide is cleaved from the support resin before addition of themaleimide. For a modification of the peptide from the C terminus end,all protecting groups are present except at the carboxy terminus (whichstays unprotected due to cleavage from the support resin) and at the twocysteines, which need to be deprotected when peptide is cleaved fromresin. Mild air oxidation yields the disulfide bridge, and the peptidecan be purified at that stage. Solution phase chemistry is then requiredto activate the C-terminus in presence of the disulfide bridge and addthe maleimide (through an amino-alkyl-maleimide) to the C-terminus. Thepeptide is then fully deprotected.

For a modification of the peptide at the N-terminus, the peptide canremain on the support resin. The two cysteines are selectivelydeprotected before addition of the maleimide. Air oxidation, potentiallyhelped by a catalyst (heterogeneous) can yield the disulfide with thepeptide still on the resin. Maleimide is then added on the N-terminusand peptide cleaved from resin and fully deprotected. The generalizedreaction scheme for modification at an internal amino acid of a peptidethat contains two cysteines in a disulfide bridge is illustrated in theschematic diagram below.

Alternatively, the peptide may be modified at an internal amino acid(i.e. neither at the C-terminus nor at the N-terminus). The generalizedreaction scheme for modification at an internal amino acid of a peptidethat contains two cysteines in a disulfide bridge is illustrated in theschematic diagram below.

Examples of therapeutic peptides that contain two cysteines as adisulfide bridge include human osteocalcin 1-49, human diabetesassociated peptide, the 5-28 fragment of human/canine atrial natriureticpeptide, bovine bactenecin, and human [Tyr0]-cortistatin 29.

D. Peptides Containing Multiple Cysteines

Where the peptide contains multiple cysteines as disulfide bridges, thepeptide is cleaved from the support resin before addition of themaleimide. For a modification of the peptide from the C terminus end,all protecting groups are present except at the carboxy terminus (whichstays unprotected due to cleavage from the support resin) and at the twocysteines that are supposed to build a disulfide bridge. Cysteines thatare involved in other disulfide bridges are deprotected sequencially inpairs using a choice of protecting groups. It is recommended to buildand purify each bridge one at a time prior to moving on to the nextbridge. Mild air oxidation yields the disulfide bridge, and the peptideshould be purified at each stage. Solution phase chemistry is thenrequired to activate the C-terminus in presence of the disulfide bridgeand add the maleimide (through an amino-alkyl-maleimide) to theC-terminus. The peptide is then fully deprotected.

For a modification of the peptide from the N terminus end, one can leavethe peptide on the support resin and selectively deprotect the first twocysteines to build the disulfide under mild air oxidation. Subsequentdeprotection will offer the other disulfides before addition of themaleimide. Air oxidation, potentially helped by a catalyst(heterogeneous) can yield the disulfides with the peptide still on theresin. Maleimide is then added on the N-terminus and peptide cleavedfrom resin and fully deprotected.

Alternatively, the peptide may be modified at an internal amino acid(i.e. neither at the C-terminus nor at the N-terminus).

Peptides containing multiple cysteines include human endothelins and[Lys4] Sarafotoxin S6c.

5. Administration of the Modified Therapeutic Peptides

The modified therapeutic peptide will be administered in aphysiologically acceptable medium, e.g. deionized water, phosphatebuffered saline (PBS), saline, aqueous ethanol or other alcohol, plasma,proteinaceous solutions, mannitol, aqueous glucose, alcohol, vegetableoil, or the like. Other additives which may be included include buffers,where the media are generally buffered at a pH in the range of about 5to 10, where the buffer will generally range in concentration from about50 to 250 mM, salt, where the concentration of salt will generally rangefrom about 5 to 500 mM, physiologically acceptable stabilizers, and thelike. The compositions may be lyophilized for convenient storage andtransport.

The modified Itherapeutic peptides will for the most part beadministered orally, parenterally, such as intravascularly (IV),intraarterially (IA), intramuscularly (IM), subcutaneously (SC), or thelike. Administration may in appropriate situations be by transfusion. Insome instances, where reaction of the functional group is relativelyslow, administration may be oral, nasal, rectal, transdermal or aerosol,where the nature of the conjugate allows for transfer to the vascularsystem. Usually a single injection will be employed although more thanone injection may be used, if desired. The modified therapeutic peptidesmay be administered by any convenient means, including syringe, trocar,catheter, or the like. The particular manner of administration will varydepending upon the amount to be administered, whether a single bolus orcontinuous administration, or the like. Preferably, the administrationwill be intravascularly, where the site of introduction is not criticalto this invention, preferably at a site where there is rapid blood flow,e.g., intravenously, peripheral or central vein. Other routes may finduse where the administration is coupled with slow release techniques ora protective matrix. The intent is that the Itherapeutic peptides beeffectively distributed in the blood, so as to be able to react with theblood components. The concentration of the conjugate will vary widely,generally ranging from about 1 pg/ml to 50 mg/ml. The total administeredintravascularly will generally be in the range of about 0.1 mg/ml toabout 10 mg/ml, more usually about 1 mg/ml to about 5 mg/ml.

By bonding to long-lived components of the blood, such asimmunoglobulin, serum albumin, red blood cells and platelets, a numberof advantages ensue. The activity of the modified therapeutic peptidescompound is extended for days to weeks. Only one administration need begiven during this period of time. Greater specificity can be achieved,since the active compound will be primarily bound to large molecules,where it is less likely to be taken up intracellularly to interfere withother physiological processes.

The formation of the covalent bond between the blood component may occurin vivo or ex vivo. For ex vivo covalent bond formation, the modifiedItherapeutic peptide is added to blood, serum or saline solutioncontaining human serum albumin or IgG to permit covalent bond formationbetween the modified therapeutic peptide and the blood component. In apreferred format, the therapeutic peptide is modified with maleimide andit is reacted with human serum albumin in saline solution. Once themodified therapeutic peptide has reacted with the blood component, toform a therapeutic peptide-protein conjugate, the conjugate may beadministered to the patient.

Alternatively, the modified therapeutic peptide may be administered tothe patient directly so that the covalent bond forms between themodified Itherapeutic peptide and the blood component in vivo.

In addition, where localized delivery of therapeutic peptides isdesired, several methods of delivery may be used:

A. Open Surgical Field Lavage

There are a number of indications for local therapeutic compounds whichwould entail administration of the therapeutic compound as an adjunct toopen surgery. In these cases, the therapeutic compound would either belavaged in the surgical site (or a portion of that site) prior toclosure, or the therapeutic compound would be incubated for a short timein a confined space (e.g., the interior of a section of an arteryfollowing an endarterectomy procedure or a portion of GI tract duringresection) and the excess fluid subsequently evacuated.

B. Incubation of Tissue Grafts

Tissue grafts such as autologous and xenobiotic vein/artery and valvegrafts as well as organ grafts can be pretreated with therapeuticcompounds that have been modified to permit covalent bond formation byeither incubating them in a therapeutic solution and/or perfusing themwith such a solution.

C. Catheter Delivery

A catheter is used to deliver the therapeutic compound either as part ofan endoscopic procedure into the interior of an organ (e.g., bladder, GItract, vagina/uterus) or adjunctive to a cardiovascular catheterprocedure such as a balloon angioplasty. Standard catheters as well asnewer drug delivery and iontophoretic catheters can be utilized.

D. Direct Injection

For certain poorly vascularized spaces such as intra-articular jointspaces, a direct injection of a therapeutic compound may be able tobioconjugate to surface tissues and achieve a desirable duration of drugeffect. Other applications could include intra medullary, intratumor,intravaginal, intrauterine, intra intestinal, intra eustachian tube,intrathecal, subcutaneous, intrarticular, intraperitoneal or intraocularinjections as weel as via bronchoscope, via nasogastiric tube and vianophrostomy.

6. Monitoring the Presence of Modified Therapeutic Peptide Derivatives

Another aspect of this invention relates to methods for determining theconcentration of the therapeutic peptides and/or analogs, or theirderivatives and conjugates in biological samples (such as blood) anddetermining the peptidase stability of the modified peptides. The bloodof the mammalian host may be monitored for the presence of the modifiedtherapeutic peptide compounds one or more times. By taking a portion orsample of the blood of the host, one may determine whether thetherapeutic peptide has become bound to the long-lived blood componentsin sufficient amount to be therapeutically active and, thereafter, thelevel of therapeutic peptide compound in the blood. If desired, one mayalso determine to which of the blood components the therapeutic peptidederivative molecule is bound. This is particularly important when usingnon-specific therapeutic peptides. For specific maleimide-therapeuticpeptides, it is much simpler to calculate the half life of serum albuminand IgG.

One method for determining the concentration of the therapeutic peptide,analogs, derivatives and conjugates is to use antibodies specific to thetherapeutic peptides or therapeutic peptide analogs or their derivativesand conjugates, and to use such antibodies as a treatment for toxicitypotentially associated with such therapeutic peptides, analogs, and/ortheir derivatives or conjugates. This is advantageous because theincreased stability and life of the therapeutic peptides in vivo in thepatient might lead to novel problems during treatment, includingincreased possibility for toxicity. It should be mentioned, however,that in some cases, the traditional antibody assay may not recognize thedifference between cleaved and uncleaved therapeutic peptides. In suchcases, other assay techniques may be employed, for example LC/MS (LiquidChromatography/Mass Spectrometry).

The use of antibodies, either monoclonal or polyclonal, havingspecificity for a particular therapeutic peptide, analog or derivativethereof, can assist in mediating any such problem. The antibody may begenerated or derived from a host immunized with the particulartherapeutic peptide, analog or derivative thereof, or with animmunogenic fragment of the agent, or a synthesized immunogencorresponding to an antigenic determinant of the agent. Preferredantibodies will have high specificity and affinity for native,derivatized and conjugated forms of the therapeutic peptide ortherapeutic peptide analog. Such antibodies can also be labeled withenzymes, fluorochromes, or radiolabels.

Antibodies specific for derivatized therapeutic peptides may be producedby using purified therapeutic peptides for the induction of derivatizedtherapeutic peptide-specific antibodies. By induction of antibodies, itis intended not only the stimulation of an immune response by injectioninto animals, but analogous steps in the production of syntheticantibodies or other specific binding molecules such as screening ofrecombinant immunoglobulin libraries. Both monoclonal and polyclonalantibodies can be produced by procedures well known in the art. In somecases, the use of monoclonal antibodies may be preferred over polyclonalantibodies, such as when degradation occurs over an area not covered byepitope/antibody recognition.

The antibodies may be used to treat toxicity induced by administrationof the therapeutic peptide, analog or derivative thereof, and may beused ex vivo or in vivo. Ex vivo methods would include immuno-dialysistreatment for toxicity employing antibodies fixed to solid supports. Invivo methods include administration of antibodies in amounts effectiveto induce clearance of antibody-agent complexes.

The antibodies may be used to remove the therapeutic peptides, analogsor derivatives thereof, and conjugates thereof, from a patient's bloodex vivo by contacting the blood with the antibodies under sterileconditions. For example, the antibodies can be fixed or otherwiseimmobilized on a column matrix and the patient's blood can be removedfrom the patient and passed over the matrix. The therapeutic peptideanalogs, derivatives or conjugates, will bind to the antibodies and theblood containing a low concentration of the therapeutic peptide, analog,derivative or conjugate, then may be returned to the patient'scirculatory system. The amount of therapeutic peptide compound removedcan be controlled by adjusting the pressure and flow rate. Preferentialremoval of the therapeutic peptides, analogs, derivatives and conjugatesfrom the plasma component of a patient's blood can be affected, forexample, by the use of a semipermeable membrane, or by otherwise firstseparating the plasma component from the cellular component by waysknown in the art prior to passing the plasma component over a matrixcontaining the anti-therapeutic antibodies. Alternatively thepreferential removal of therapeutic peptide-conjugated blood cells,including red blood cells, can be effected by collecting andconcentrating the blood cells in the patient's blood and contactingthose cells with fixed anti-therapeutic antibodies to the exclusion ofthe serum component of the patient's blood.

The antibodies can be administered in vivo, parenterally, to a patientthat has received the therapeutic peptide, analogs, derivatives orconjugates for treatment. The antibodies will bind the therapeuticpeptide compounds and conjugates. Once bound the therapeutic peptide,activity will be hindered if not completely blocked thereby reducing thebiologically effective concentration of therapeutic peptide compound inthe patient's bloodstream and minimizing harmful side effects. Inaddition, the bound antibody-therapeutic peptide complex will facilitateclearance of the therapeutic peptide compounds and conjugates from thepatient's blood stream.

The invention having been fully described is now exemplified by thefollowing non-limiting examples.

EXAMPLES

A. General Method of Synthesis of a Modified Therapeutic Peptide

Solid phase peptide synthesis of the modified peptide on a 100 μmolescale was performed on a Symphony Peptide Synthesizer using Fmocprotected Rink Amide MBHA resin, Fmoc protected amino acids,O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) in N,N-dimethylformamide (DMF) solution and activation withN-methyl morpholine (NMM), and piperidine deprotection of Fmoc groups(Step 1). The deprotection of the terminal Fmoc group is accomplishedusing 20% piperidine (Step 2) followed by either the coupling of3-maleimidopropionic acid (3-MPA), the coupling of acetic acid or thecoupling of one or multiple Fmoc-AEEA followed by the coupling of 3-MPA(Step 3). Resin cleavage and products isolation are performed using 86%TFA/5% TIS/5% H₂O/2% thioanisole and 2% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The products are purifiedby preparative reverse phase HPLC using a Varian (Rainin) preparativebinary HPLC system using a Dynamax C₁₈, 60 Å, 8 μm, 21 mm×25 cm columnequipped with a Dynamax C₁₈, 60 Å, 8 μm guard module, 21 mm×25 cm columnand UV detector (Varian Dynamax UVD II) at λ 214 and 254 nm. The productshould have >95% purity as determined by RP-HPLC mass spectrometry usinga Hewlett Packard LCMS-1100 series spectrometer equipped with a diodearray detector and using electro-spray ionization.

B. Alteration of the Native Peptide Chain

To facilitate modification of the peptide, one or more amino acidresidues may be added to the peptide as described in examples 1 to 5,and/or one or more amino acid residues may be replaced with other aminoacid residues. This alteration aids attachment of the reactive group.

Example 1

Addition of Lys at C-Terminus of Kringle-5

Preparation of NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-NH₂.3TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH. Deblocking of the Fmocgroup the the N-terminal of the resin-bound amino acid was performedwith 20% piperidine in DMF for about 15-20 minutes. Coupling of theacetic acid was performed under conditions similar to amino acidcoupling. Final cleavage from the resin was performed using cleavagemixture as described above. The product was isolated by precipitationand purified by preparative HPLC to afford the desired product as awhite solid upon lyophilization.

Example 2

Addition of Lys at C-Terminus of Kringle-5

Preparation of NAc-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-NH₂.2TFA.3TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH. Deblocking of the Fmoc group the theN-terminal of the resin-bound amino acid was performed with 20%piperidine in DMF for about 15-20 minutes. Coupling of the acetic acidwas performed under conditions similar to amino acid coupling. Finalcleavage from the resin was performed using cleavage mixture asdescribed above. The product was isolated by precipitation and purifiedby preparative HPLC to afford the desired product as a white solid uponlyophilization.

Example 3

Addition of Lys at N-Terminus of Kringle-5

Preparation ofNAc-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-NH₂.3TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)OH. Deblocking of theFmoc group the the N-terminal of the resin-bound amino acid wasperformed with 20% piperidine in DMF for about 15-20 minutes. Couplingof the acetic acid was performed under conditions similar to amino acidcoupling. Final cleavage from the resin was performed using cleavagemixture as described above. The product was isolated by precipitationand purified by preparative HPLC to afford the desired product as awhite solid upon lyophilization.

Example 4

Addition of Lys at N-Terminus of Kringle-5, Substitution of Cys with Alaat Position 524

Preparation ofNAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-Ala⁵²⁴-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-NH₂.4TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Ala-OH,Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH,Fmoc-Arg(Pbf)-OH. Deblocking of the Fmoc group the the N-terminal of theresin-bound amino acid was performed with 20% piperidine in DMF forabout 15-20 minutes. Coupling of the acetic acid was performed underconditions similar to amino acid coupling. Final cleavage from the resinwas performed using cleavage mixture as described above. The product wasisolated by precipitation and purified by preparative HPLC to afford thedesired product as a white solid upon lyophilization.

Example 5

Addition of Lys at N-Terminus of Kringle-5

Preparation ofNAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-Lys-NH₂.2TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH,Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH. Deblocking of the Fmoc group the theN-terminal of the resin-bound amino acid was performed with 20%piperidine in DMF for about 15-20 minutes. Coupling of the acetic acidwas performed under conditions similar to amino acid coupling. Finalcleavage from the resin was performed using cleavage mixture asdescribed above. The product was isolated by precipitation and purifiedby preparative HPLC to afford the desired product as a white solid uponlyophilization.

Example 6

Preparation of D-Ala2 GLP-1 (7-46) Amide

Solid phase peptide synthesis of the GLP-1 analog on a 100 μmole scaleis performed using manual solid-phase synthesis and a Symphony PeptideSynthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc protectedamino acids, O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF) solution andactivation with N-methyl morpholine (NMM), and piperidine deprotectionof Fmoc groups (Step 1). Resin cleavage and product isolation isperformed using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 2). The product is purified bypreparative reversed phased HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desiredpeptide in >95% purity, as determined by RP-HPLC. These steps areillustrated in the schematic diagram below.

C. Preparation of Modified Peptides from Peptides Containing NoCysteines

Preparation of maleimido peptides from therapeutic peptides containingmultiple protected functional groups and no Cysteine is exemplified bythe synthesis of peptides as described below. The peptide may bemodified at the N-terminus, the C-terminus, or at an amino acid locatedbetween the N-terminus and the C-terminus. The modified peptide issynthesized by linking off the N-terminus of the natural peptidesequence or by linking off the modified C-terminus of the naturalpeptide sequence. One or more additional amino acids may be added to thetherapeutic peptide to facilitate attachment of the reactive group.

1. Modification of the Therapeutic Peptide at the C-Terminus

Example 7

Modification of RSV Peptide at the ε-Amino Group of the Added C-terminusLysine Residue

Preparation ofVal-Ile-Thr-Ile-Glu-Leu-Ser-Asn-Ile-Lys-Glu-Asn-Lys-Met-Asn-Gly-Ala-Lys-Val-Lys-Leu-Ile-Lys-Gln-Glu-Leu-Asp-Lys-Tyr-Lys-Asn-Ala-Val-Lys-(Nε-MPA)

Solid phase peptide synthesis of the DAC analog on a 100 μmole scale isperformed using manual solid-phase synthesis, a Symphony PeptideSynthesizer and Fmoc protected Rink Amide MBHA. The following protectedamino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH,Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH,Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH,Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH,Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Met-OH,Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH,Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH,Fmoc-Val-OH. They are dissolved in N,N-dimethylformamide (DMF) and,according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup is achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (step 1). The selectivedeprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh3)4dissolved in 5 mL of CHCl3:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Theresin is then washed with CHCl3 (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM(6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for theaddition of the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin is washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide is cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et2O (Step 4). The product is purified bypreparative reverse phase HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H2O (A)and 0.045% TFA in CH3CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desired DACin >95% purity, as determined by RP-HPLC.

Example 8

Modification of Dyn A 1-13 at the ε-Amino Group of the Added C-terminusLysine Residue—Synthesis of Dyn A 1-13(Nε-MPA)-NH₂

Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-(Nε-MPA)-NH₂

Solid phase peptide synthesis of a modified Dyn A 1-13 on a 100 μmolescale was performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Rink Amide MBHA. The followingprotected amino acids were sequentially added to resin:Fmoc-Lys(Aloc)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH.They were dissolved in N,N-dimethylformamide (DMF) and, according to thesequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup is achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (Step 1). The selectivedeprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Theresin is then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM(6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for theaddition of the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin is washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide is cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The product is purified bypreparative reverse phase HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desired DACin >95% purity, as determined by RP-HPLC.

The structure of this product is

Example 9

Modification of Dyn A 2-13 at the ε-Amino Group of the Added C-terminusLysine Residue—Synthesis of Dyn A 2-13(Nε-MPA)-NH₂Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-(Nε-MPA)-NH₂

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Mtt)-OH,Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH,Fmoc-Phe-OH, Fmoc-Gly-OH, and Boc-Gly-OH. Manual synthesis was employedfor the remaining steps: selective removal of the Mtt group and couplingof MPA using HBTU/HOBt/DIEA activation in DMF. The target dynorphinanalog was removed from the resin; the product was isolated byprecipitation and purified by preparative HPLC to afford the desiredproduct as a white solid upon lyophilization in a 35% yield. Anal. HPLCindicated product to be >95% pure with R_(t)=30.42 min. ESI-MS m/z forC₇₃H₁₂₃N₂₅O₁₅ (MH⁺), calcd 1590.0, found MH³⁺ 531.3.

Example 10

Modification of Dyn A 1-13 at the ε-Amino Group of the Added C-terminusLysine Residue—Synthesis of Dyn A 1-13(AEA₃-MPA)-NH₂Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-(AEA₃-MPA)-NH₂

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Mtt)-OH,Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH,Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, and Boc-Tyr(Boc)-OH. Manualsynthesis was employed for the remaining steps: selective removal of theMtt group, the coupling of three-Fmoc-AEA-OH groups(AEA=aminoethoxyacetic acid) with Fmoc removal in-between each coupling,and MPA acid using HBTU/HOBt/DIEA activation in DMF. The targetdynorphin analog was removed from the resin; the product was isolated byprecipitation and purified by preparative HPLC to afford the desiredproduct as a white solid upon lyophilization in a 29% yield. Anal. HPLCindicated product to be >95% pure with R_(t)=33.06 min. ESI-MS m/z forC₉₄H₁₅₄N₂₉O₂₃ (MH⁺), calcd 2057.2, found MH⁴⁺ 515.4, MH³⁺ 686.9, MH²⁺1029.7.

Example 11

Modification of Dyn A 2-13 at the ε-Amino Group of the Added C-terminusLysine Residue—Synthesis of Dyn A 2-13(AEA₃-MPA)-NH₂Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-(AEA₃-MPA)-NH₂

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Mtt)-OH,Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH,Fmoc-Phe-OH, Fmoc-Gly-OH, and Fmoc-Gly-OH. Manual synthesis was employedfor the remaining steps: selective removal of the Mtt group, thecoupling of three-Fmoc-AEA-OH groups, with Fmoc removal in-between eachcoupling, and MPA using HBTU/HOBt/DIEA activation in DMF. The targetdynorphin analog was removed from the resin; the product was isolated byprecipitation and purified by preparative HPLC to afford the desiredproduct as a white solid upon lyophilization in a 29% yield. Anal. HPLCindicated product to be >95% pure with R_(t)=31.88 min. ESI-MS m/z forC₈₅H₁₄₅N₂₅O₂₁ (MH⁺), calcd 1894.3, found MH⁴⁺ 474.6, MH³⁺ 632.4, MH²⁺948.10.

Example 12

Modification of Neuropeptide Y at the ε-Amino Group of the AddedC-terminus Lysine Residue

Preparation ofTyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Met-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Lys-(N-εMPA)-NH₂

Solid phase peptide synthesis of a modified neuropeptide Y analog on a100 μmole scale is performed using manual solid-phase synthesis, aSymphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. Thefollowing protected amino acids were sequentially added to resin:Fmoc-Lys(Aloc)-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,Fmoc-Met-OH, Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH,Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(tBu)-OH,Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH,Fmoc-Tyr(tBu)-OH. They are dissolved in N,N-dimethylformamide (DMF) and,according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup is achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (Step 1). The selectivedeprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Theresin is then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM(6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for theaddition of the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin is washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide is cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The product is purified bypreparative reverse phase HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desired DACin >95% purity, as determined by RP-HPLC.

Example 13

Modification of GLP-1 (7-36) at the C-Terminus Arginine

Preparation of GLP-1 (7-36)-EDA-MPA

Solid phase peptide synthesis of a modified GLP-1 analog on a 100 μmolescale is performed manually and on a Symphony Peptide Synthesizer SASRIN(super acid sensitive resin). The following protected amino acids aresequentially added to the resin: Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH,Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH,Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH,Fmoc-Gly-OH, Fmco-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(tBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Boc-His(Trt)-OH. They aredissolved in N,N-dimethylformamide (DMF) and, according to the sequence,activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group is achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (Step 1). Thefully protected peptide is cleaved from the resin by treatment with 1%TFA/DCM (Step 2). Ethylenediamine and 3-maleimidopropionic acid are thensequentially added to the free C-terminus (Step 3). The protectinggroups are then cleaved and the product isolated using 86% TFA/5% TIS/5%H₂O/2% thioanisole and 2% phenol, followed by precipitation by dry-icecold Et₂O (Step 4). The product is purified by preparative reverse phaseHPLC using a Varian (Rainin) preparative binary HPLC system using aDynamax C₁₈, 60 Å, 8 μm, 21 mm×25 cm column equipped with a Dynamax C₁₈,60 Å, 8 μm guard module, 21 mm×25 cm column and UV detector (VarianDynamax UVD II) at λ 214 and 254 nm to afford the desired DAC in >95%purity, as determined by RP-HPLC. These steps are illustrated in theschematic diagram below.

Example 14

Modification of Exendin-4 at the C-terminus Serine

Preparation of Exendin-4 (1-39)-EDA-MPA

Solid phase peptide synthesis of a modified Exendin-4 analog on a 100μmole scale is performed manually and on a Symphony Peptide SynthesizerSASRIN (super acid sensitive resin). The following protected amino acidsare sequentially added to the resin: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH,Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH,Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Leu-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Boc-His(Trt)-OH. They aredissolved in N,N-dimethylformamide (DMF) and, according to the sequence,activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group is achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (Step 1). Thefully protected peptide is cleaved from the resin by treatment with 1%TFA/DCM (Step 2). Ethylenediamine and 3-maleimidopropionic acid are thensequentially added to the free C-terminus (Step 3). The protectinggroups are then cleaved and the product isolated using 86% TFA/5% TIS/5%H₂O/2% thioanisole and 2% phenol, followed by precipitation by dry-icecold Et₂O (Step 4). The product is purified by preparative reverse phaseHPLC using a Varian (Rainin) preparative binary HPLC system using aDynamax C₁₈, 60 Å, 8 μm, 21 mm×25 cm column equipped with a Dynamax C₁₈,60 Å, 8 μm guard module, 21 mm×25 cm column and UV detector (VarianDynamax UVD II) at λ 214 and 254 nm to afford the desired DAC in >95%purity, as determined by RP-HPLC.

Example 15

Modification of Secretin Peptide at the ε-Amino Group of the AddedC-terminus Lysine Residue

Preparation ofHis-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-Glu-Gly-Ala-Arg-Leu-Glu-Arg-Leu-Leu-Gln-Gly-Leu-Val-Lys-(Nε-MPA)-NH₂

Solid phase peptide synthesis of a modified secretin peptide analog on a100 μmole scale is performed using manual solid-phase synthesis, aSymphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. Thefollowing protected amino acids are sequentially added to resin:Fmoc-Lys(Aloc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Gly-OH,Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Gly-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-His(Boc)-OH. Theyare dissolved in N,N-dimethylformamide (DMF) and, according to thesequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup is achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (Step 1). The selectivedeprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Theresin is then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM(6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for theaddition of the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin is washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide is cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The product is purified bypreparative reverse phase HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desired DACin >95% purity, as determined by RP-HPLC.

Example 16

Modification of Kringle-5 at the ε-Amino Group of the Added C-terminusLysine Residue

Preparation of NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-(Nε-MPA)-NH₂.2TFA

Solid phase peptide synthesis of a modified Kringle-5 peptide on a 100μmole scale was performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Rink Amide MBHA. The followingprotected amino acids are sequentially added to resin:Fmoc-Lys(Aloc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH. They weredissolved in N,N-dimethylformamide (DMF) and, according to the sequence,activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group is achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). Atthe end of the synthesis Acetic Anhydride was added to acetylate theN-terminal. The selective deprotection of the Lys (Aloc) group isperformed manually and accomplished by treating the resin with asolution of 3 eq of Pd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc(18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃ (6×5mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). Thesynthesis is then re-automated for the addition of the3-maleimidopropionic acid (Step 3). Between every coupling, the resin iswashed 3 times with N,N-dimethylformamide (DMF) and 3 times withisopropanol. The peptide is cleaved from the resin using 85% TFA/5%TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-icecold Et₂O (Step 4). The product is purified by preparative reverse phaseHPLC using a Varian (Rainin) preparative binary HPLC system: gradientelution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B))over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ 214 and 254nm to afford the desired DAC in >95% purity, as determined by RP-HPLC.

Example 17

Modification of Kringle-5 at the ε-Amino Group of the Added C-terminusLysine Residue

Preparation ofNAc-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-(Nε-MPA)-NH₂.2TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)OH (step 1). Deblockingof the Fmoc group the the N-terminal of the resin-bound amino acid wasperformed with 20% piperidine in DMF for about 15-20 minutes. Finalcleavage from the resin was performed using cleavage mixture asdescribed above. The product was isolated by precipitation and purifiedby preparative HPLC to afford the desired product as a white solid uponlyophilization The selective deprotection of the Lys(Aloc) group wasperformed manually and accomplished by treating the resin with asolution of 3 eq of Pd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc(18:1:0.5) for 2 h (Step 2). The resin was then washed with CHCl₃ (6×5mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). Thesynthesis was then re-automated for the addition of the3-maleimidopropionic acid (Step 3). Resin cleavage and product isolationwas performed using 85% TFA/5% TIS/5% thioanisole and 5% phenol,followed by precipitation by dry-ice cold Et₂O (Step 4). The product waspurified by preparative reversed phased HPLC using a Varian (Rainin)preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFAin H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min usinga Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm.

Example 18

Modification of Kringle-5 at the ε-Amino Group of the Added C-terminusLysine Residue

Preparation ofNAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-Ala-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-(Nε-MPA)-NH₂.3TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Ala-OH,Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH,Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH (step 1). Deblocking of the Fmocgroup the the N-terminal of the resin-bound amino acid was performedwith 20% piperidine in DMF for about 15-20 minutes. Coupling of theacetic acid was performed under conditions similar to amino acidcoupling. Final cleavage from the resin was performed using cleavagemixture as described above. The product was isolated by precipitationand purified by preparative HPLC to afford the desired product as awhite solid upon lyophilization.

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the 3-maleimidopropionic acid (Step 3). Resincleavage and product isolation was performed using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product was purified by preparative reversed phasedHPLC using a Varian (Rainin) preparative binary HPLC system: gradientelution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B))over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ 214 and 254nm.

Example 19

Modification of Kringle-5 at the ε-Amino Group of the Added C-terminusLysine Residue

Preparation ofNAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-Lys-(Nε-MPA)-NH₂.TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH,Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH (Step 1). The selective deprotectionof the Lys(Aloc) group was performed manually and accomplished bytreating the resin with a solution of 3 eq of Pd(PPh₃)₄ dissolved in 5mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin was thenwashed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), andDMF (6×5 mL). The synthesis was then re-automated for the addition ofthe 3-maleimidopropionic acid (Step 3). Resin cleavage and productisolation was performed using 85% TFA/5% TIS/5% thioanisole and 5%phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct was purified by preparative reversed phased HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) at λ 214 and 254 nm.

Example 20

Modification of Kringle-5 at the ε-Amino Group of the Added C-terminusLysine Residue

Preparation of NAc-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-(Nε-MPA)-NH₂.2TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH (Step 1). Deblocking of the Fmocgroup the N-terminal of the resin-bound amino acid was performed with20% piperidine in DMF for about 15-20 minutes. Coupling of the aceticacid was performed under conditions similar to amino acid coupling.Final cleavage from the resin was performed using cleavage mixture asdescribed above. The product was isolated by precipitation and purifiedby preparative HPLC to afford the desired product as a white solid uponlyophilization The selective deprotection of the Lys(Aloc) group wasperformed manually and accomplished by treating the resin with asolution of 3 eq of Pd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc(18:1:0.5) for 2 h (Step 2). The resin was then washed with CHCl₃ (6×5mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). Thesynthesis was then re-automated for the addition of the3-maleimidopropionic acid (Step 3). Resin cleavage and product isolationwas performed using 85% TFA/5% TIS/5% thioanisole and 5% phenol,followed by precipitation by dry-ice cold Et₂O (Step 4). The product waspurified by preparative reversed phased HPLC using a Varian (Rainin)preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFAin H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min usinga Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm.

Example 21

Modification of Kringle-5 at the ε-Amino Group of the Added C-terminusLysine Residue

Preparation of NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Lys-(Nε-MPA)-NH₂.2TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH (Step 1). Deblocking of the Fmoc group theN-terminal of the resin-bound amino acid was performed with 20%piperidine in DMF for about 15-20 minutes. Coupling of the acetic acidwas performed under conditions similar to amino acid coupling. Finalcleavage from the resin was performed using cleavage mixture asdescribed above. The product was isolated by precipitation and purifiedby preparative HPLC to afford the desired product as a white solid uponlyophilization

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the 3-maleimidopropionic acid (Step 3). Resincleavage and product isolation was performed using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product was purified by preparative reversed phasedHPLC using a Varian (Rainin) preparative binary HPLC system: gradientelution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B))over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ 214 and 254nm.

Example 22

Modification of Kringle-5 at the ε-Amino Group of the Added C-terminusLysine Residue

Preparation ofNAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-(Nε-AEEA-MPA)-NH₂.2TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH (Step 1). Deblocking ofthe Fmoc group at the N-terminal of the resin-bound amino acid wasperformed with 20% piperidine in DMF for about 15-20 minutes. Couplingof the acetic acid was performed under conditions similar to amino acidcoupling. The selective deprotection of the Lys(Aloc) group wasperformed manually and accomplished by treating the resin with asolution of 3 eq of Pd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc(18:1:0.5) for 2 h (Step 2). The resin was then washed with CHCl₃ (6×5mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). Thesynthesis was then re-automated for the addition. The synthesis was thenre-automated for the addition of the AEEA (aminoethoxyethoxyacetic acid)group and of the 3-maleimidopropionic acid (MPA) (Step 3). Resincleavage and product isolation was performed using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product was purified by preparative reversed phasedHPLC using a Varian (Rainin) preparative binary HPLC system: gradientelution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B))over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ 214 and 254nm.

Example 23

Modification of Kringle-5 at the ε-Amino Group of the Added C-terminusLysine Residue

Preparation ofNAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-(Nε-AEEA_(n)-MPA)-NH₂.2TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH (Step 1). Deblocking ofthe Fmoc group at the N-terminal of the resin-bound amino acid wasperformed with 20% piperidine in DMF for about 15-20 minutes. Couplingof the acetic acid was performed under conditions similar to amino acidcoupling.

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition. The synthesis was then re-automated for the additionof n AEEA (aminoethoxyethoxyacetic acid) groups and of the3-maleimidopropionic acid (MPA) (Step 3). Resin cleavage and productisolation was performed using 85% TFA/5% TIS/5% thioanisole and 5%phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct was purified by preparative reversed phased HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) at λ 214 and 254 nm.

Example 24

Modification of GLP-1 at the ε-Amino Group of the Added C-terminusLysine Residue

Preparation of GLP-1 (1-36)-Lys³⁷(Nε-MPA)-NH₂.5TFA;His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys(Nε-MPA)-NH₂.5TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH,Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH,Fmoc-Tyr(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH,Boc-His(N-Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Boc-His(N-Trt)-OH (step 1)

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the 3-maleimidopropionic acid (Step 3). Resincleavage and product isolation was performed using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product was purified by preparative reverse phaseHPLC using a Varian (Rainin) preparative binary HPLC system: gradientelution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B))over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ 214 and 254nm. The product had >95% purity as determined by RP-HPLC massspectrometry using a Hewlett Packard LCMS-1100 series spectrometerequipped with a diode array detector and using electro-spray ionization.These steps are illustrated in the schematic diagram below.

Example 25

Modification of GLP-1 at the ε-Amino Group of the Added C-terminusLysine Residue

Preparation of GLP-1 (1-36)-Lys³⁷(Nε-AEEA-AEEA-MPA)-NH₂.5TFA;His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys(Nε-AEEA-AEEA-MPA)-NH₂.5TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH,Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH,Fmoc-Tyr(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH,Boc-His(N-Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Boc-His(N-Trt)-OH (step 1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the two AEEA (aminoethoxyethoxyacetic acid) groupsand the 3-maleimidopropionic acid (Step 3). Resin cleavage and productisolation was performed using 85% TFA/5% TIS/5% thioanisole and 5%phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct was purified by preparative reverse phase HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) at λ 214 and 254 nm. The producthad >95% purity as determined by RP-HPLC mass spectrometry using aHewlett Packard LCMS-1100 series spectrometer equipped with a diodearray detector and using electro-spray ionization, ESI-MS m/z forC₁₇₄H₂₆₅N₄₄O₅₆ (MH⁺), calcd 3868, found [M+H₂]²⁺ 1934, [M+H₃]³⁺ 1290,[M+H₄]⁴⁺ 967. These steps are illustrated in the schematic diagrambelow.

Example 26

Modification of GLP-1 at the ε-Amino Group of the Added C-terminusLysine Residue

Preparation of GLP-1 (7-36)-Lys³⁷(Nε-MPA)-NH₂.4TFA;His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys(Nε-MPA)-NH₂.4TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH,Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH,Fmoc-Tyr(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH,Boc-His(N-Trt)-OH (Step 1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the 3-maleimidopropionic acid (Step 3). Resincleavage and product isolation was performed using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product was purified by preparative reverse phaseHPLC using a Varian (Rainin) preparative binary HPLC system: gradientelution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B))over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ 214 and 254nm. The product had >95% purity as determined by RP-HPLC massspectrometry using a Hewlett Packard LCMS-1100 series spectrometerequipped with a diode array detector and using electro-spray ionization.

Example 27

Modification of GLP-1 at the ε-Amino Group of the Added C-terminusLysine Residue

Preparation of GLP-1 (7-36)-Lys³⁷(Nε-AEEA-AEEA-MPA)-NH₂.4TFAHis-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys(Nε-AEEA-AEEA-MPA)-NH₂.4TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH,Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH,Fmoc-Tyr(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH,Boc-His(N-Trt)-OH (Step 1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the two AEEA (aminoethoxyethoxyacetic acid) groupsand the 3-maleimidopropionic acid (Step 3). Resin cleavage and productisolation was performed using 85% TFA/5% TIS/5% thioanisole and 5%phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct was purified by preparative reverse phase HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) at λ 214 and 254 nm. The producthad >95% purity as determined by RP-HPLC mass spectrometry using aHewlett Packard LCMS-1100 series spectrometer equipped with a diodearray detector and using electro-spray ionization.

Example 28

Modification of D-Ala² GLP-1 at the ε-Amino Group of the AddedC-terminus Lysine Residue

Preparation of D-Ala² GLP-1 (7-36)-Lys³⁷(Nε-MPA)-NH₂.4TFAHis-d-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys(Nε-MPA)-NHh₂.4TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH,Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH,Fmoc-Tyr(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-d-Ala-OH,Boc-His(N-Trt)-OH (Step 1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the 3-maleimidopropionic acid (Step 3). Resincleavage and product isolation was performed using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product was purified by preparative reverse phaseHPLC using a Varian (Rainin) preparative binary HPLC system: gradientelution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B))over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ 214 and 254nm. The product had >95% purity as determined by RP-HPLC massspectrometry using a Hewlett Packard LCMS-1100 series spectrometerequipped with a diode array detector and using electro-spray ionization.These steps are illustrated in the schematic diagram below.

Example 29

Modification of D-Ala² GLP-1 at the ε-Amino Group of the AddedC-terminus Lysine Residue

Preparation of D-Ala² GLP-1 (7-36)-Lys³⁷(Nε-AEEA-AEEA-MPA)-NH₂.4TFAHis-D-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys(Nε-AEEA-AEEA-MPA)-NH₂.4TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH,Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH,Fmoc-Tyr(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-d-Ala-OH,Boc-His(N-Trt)-OH (Step 1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the two AEEA (aminoethoxyethoxyacetic acid) groupsand the 3-maleimidopropionic acid (Step 3). Resin cleavage and productisolation was performed using 85% TFA/5% TIS/5% thioanisole and 5%phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct was purified by preparative reverse phase HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) λ 214 and 254 nm. The producthad >95% purity as determined by RP-HPLC mass spectrometry using aHewlett Packard LCMS-1100 series spectrometer equipped with a diodearray detector and using electro-spray ionization. These steps areillustrated in the schematic diagram below.

Example 30

Modification of Exendin-4(1-39) at the ε-Amino Group of the AddedC-terminus Lysine Residue

Preparation of Exendin-4 (1-39)-Lys⁴⁰(Nε-MPA)-NH₂;His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(Nε-MPA)-NH₂.5TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH,Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH,Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH,Fmoc-Trp-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Leu-OH,Fmoc-Arg(Bpf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Boc-His(Trt)-OH (Step 1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the 3-maleimidopropionic acid (Step 3). Resincleavage and product isolation was performed using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product was purified by preparative reverse phaseHPLC using a Varian (Rainin) preparative binary HPLC system: gradientelution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B))over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ 214 and 254nm. The product had >95% purity as determined by RP-HPLC massspectrometry using a Hewlett Packard LCMS-1100 series spectrometerequipped with a diode array detector and using electro-spray ionization.These steps are illustrated in the schematic diagram below.

Example 31

Modification of Exendin-4(1-39) at the ε-Amino Group of the AddedC-terminus Lysine Residue

Preparation of Exendin-4 (1-39)-Lys⁴⁰(Nε-AEEA-AEEA-MPA)-NH₂.5TFA;His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(Nε-AEEA-AEEA-MPA)-NH₂.5TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH,Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH,Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH,Fmoc-Trp-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Leu-OH,Fmoc-Arg(Bpf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Boc-His(Trt)-OH (Step 1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the two AEEA (aminoethoxyethoxyacetic acid) groupsand the 3-maleimidopropionic acid (Step 3). Resin cleavage and productisolation was performed using 85% TFA/5% TIS/5% thioanisole and 5%phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct was purified by preparative reverse phase HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) at λ 214 and 254 nm. The producthad >95% purity as determined by RP-HPLC mass spectrometry using aHewlett Packard LCMS-1100 series spectrometer equipped with a diodearray detector and using electro-spray ionization. These steps areillustrated in the schematic diagram below.

Example 32

Modification of Exendin-3(1-39) at the ε-Amino Group of the AddedC-terminus Lysine Residue

Preparation of Exendin-3 (1-39)-Lys⁴⁰(Nε-MPA)NH₂.5TFA;His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(Nε-MPA)NH₂.5TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH,Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH,Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH,Fmoc-Trp-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Leu-OH,Fmoc-Arg(Bpf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(OtBu)-OH, Boc-His(Trt)-OH (Step1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAC (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis was then re-automatedfor the addition of the 3-maleimidopropionic acid (Step 3). Resincleavage and product isolation was performed using 85% TFA/5% TIS/5%thioanisole and 5% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product was purified by preparative reverse phaseHPLC using a Varian (Rainin) preparative binary HPLC system: gradientelution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B))over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ 214 and 254nm. The product had >95% purity as determined by RP-HPLC massspectrometry using a Hewlett Packard LCMS-1100 series spectrometerequipped with a diode array detector and using electro-spray ionization.These steps are illustrated in the schematic diagram below.

Example 33

Modification of Exendin-3(1-39) at the ε-Amino Group of the AddedC-terminus Lysine Residue

Preparation of Exendin-3 (1-39)-Lys⁴⁰(Nε-AEEA-AEEA-MPA)-NH₂.5TFA;His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Lys(Nε-AEEA-AEEA-MPA)-NH₂.5TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH,Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH,Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH,Fmoc-Trp-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Leu-OH,Fmoc-Arg(Bpf)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(OtBu)-OH, Boc-His(Trt)-OH (Step1).

The selective deprotection of the Lys(Aloc) group was performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step2). The resin was then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL. The synthesis was then re-automatedfor the addition of the two AEEA (aminoethoxyethoxyacetic acid) groupsand the 3-maleimidopropionic acid (Step 3). Resin cleavage and productisolation was performed using 85% TFA/5% TIS/5% thioanisole and 5%phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct was purified by preparative reverse phase HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) at λ 214 and 254 nm. The producthad >95% purity as determined by RP-HPLC mass spectrometry using aHewlett Packard LCMS-1100 series spectrometer equipped with a diodearray detector and using electro-spray ionization.

Example 34

Modification of HIV-1 DP 178 at the C-Terminus

Preparation of Modified HIV-1 DP 178 antifusogenic PeptideTyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu-Ser-Gln-Asn-Glu-Glu-Glu-Lys-Asn-Glu-Glu-Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn-Trp-Phe-Lys-(Nε-MPA)-NH₂

Using automated peptide synthesis, the following protected amino acidsare sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Mtt)-OH,Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Trp(Boc)-OH,Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(Thu)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH; Fmoc-Gln(Trt)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH,Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH,Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH and Boc-Tyr(tBu)-OH.Manual synthesis is employed for the remaining steps: selective removalof the Mtt group and coupling of maleimidopropionic acid (MPA) usingHBTU/HOBt/DIEA activation in DMF. The target molecule is removed fromthe resin; the product is isolated by precipitation and purified bypreparative HPLC to afford the desired product as a white solid uponlyophilization.

Example 35

Modification of HIV-1 DP 107 at the C-Terminus

Preparation of Modified HIV-1 DP 107 Antifusogenic PeptideAsn-Asn-Leu-Leu-Arg-Ala-Ile-Glu-Ala-Glu-Glu-His-Leu-Leu-Glu-Leu-Thr-Val-Trp-Glu-Ile-Lys-Glu-Leu-Glu-Ala-Arg-Ile-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Glu-Lys-(Nε-MPA)NH₂

Using automated peptide synthesis, the following protected amino acidsare sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Mtt)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Val-OH,Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Val-OH,Fmoc-Thr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH,Fmoc-Asn(Trt)-OH. Manual synthesis is employed for the remaining steps:selective removal of the Mtt group and coupling of maleimidopropionicacid (MPA) using HBTU/HOBt/DIEA activation in DMF. The target analog isremoved from the resin; the product is isolated by precipitation andpurified by preparative HPLC to afford the desired product as a whitesolid upon lyophilization.

2. Modification of the Therapeutic Peptide at the N-Terminus

Example 36

Modification of RSV Peptide at the ε-Amino Group of the Added N-terminusLysine Residue

Preparation of(Nε-MPA)-Lys-Val-Ile-Thr-Ile-Glu-Leu-Ser-Asn-Ile-Lys-Glu-Asn-Lys-Met-Asn-Gly-Ala-Lys-Val-Lys-Leu-Ile-Lys-Gln-Glu-Leu-Asp-Lys-Tyr-Lys-Asn-Ala-Val

Solid phase peptide synthesis of a modified RSV peptide on a 100 μmolescale is performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Rink Amide MBHA. The followingprotected amino adds are sequentially added to resin: Fmoc-Val-OH,Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH,Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH,Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,Fmoc-Ile-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Val-OH,Fmoc-Lys(Aloc)-OH. They are dissolved in N,N-dimethylformamide (DMF)and, according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup is achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (Step 1). The selectivedeprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh3)4dissolved in 5 mL of CHCl3:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Theresin is then washed with CHCl3 (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM(6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for theaddition of the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin is washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide is cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et2O (Step 4). The product is purified bypreparative reversed phased HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H2O (A)and 0.045% TFA in CH3CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desired DACin >95% purity, as determined by RP-HPLC.

Example 37

Modification of Neuropeptide Y at the ε-Amino Group of the AddedN-terminus Lysine Residue

Preparation of(Nε-MPA)-Lys-Tyr-Pro-Ser-Lys-Pro-Glu-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Met-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu

Solid phase peptide synthesis of a modified neuropeptide Y on a 100μmole scale was performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Rink Amide MBHA. The followingprotected amino acids are sequentially added to resin: Fmoc-Leu-OH,Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Met-OH, Fmoc-Asp(tBu)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Ala-OH,Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH, Fmoc-Pro-OH,Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Aloc)-OH. Theyare dissolved in N,N-dimethylformamide (DMF) and, according to thesequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup is achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (Step 1). The selectivedeprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Theresin is then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM(6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for theaddition of the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin is washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide is cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The product is purified bypreparative reverse phase HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desired DACin >95% purity, as determined by RP-HPLC.

Example 38

Modification of Neuropeptide Y at the ε-Amino Group of the AddedN-terminus Lysine Residue

Preparation of(N-εMPA)-Lys-Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Met-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu

Solid phase peptide synthesis of a modified neuropeptide Y on a 100μmole scale was performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Rink Amide MBHA. The followingprotected amino acids are sequentially added to resin: Fmoc-Leu-OH,Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Met-OH, Fmoc-Asp(tBu)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Ala-OH,Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gly-OH, Fmoc-Pro-OH,Fmoc-Asn(Trt)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Aloc)-OH. Theyare dissolved in N,N-dimethylformamide (DMF) and, according to thesequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup is achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (Step 1). The selectivedeprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)4dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Theresin is then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM(6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for theaddition of the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin is washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide is cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et2O (Step 4). The product is purified bypreparative reverse phase HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desired DACin >95% purity, as determined by RP-HPLC.

Example 39

Modification of Dyn A 1-13 at the ε-Amino Group of the Added N-terminusLysine Residue—Synthesis of (Nε-MPA)-Dyn A 1-13-NH₂

(Nε-MPA)-Lys-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu

Solid phase peptide synthesis of a modified Dyn A 1-13 analog on a 100μmole scale is performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Rink Amide MBHA. The followingprotected amino acids are sequentially added to resin: Fmoc-Lys(Boc)-OH,Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH,Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Lys(Aloc)-OH. They were dissolved in N,N-dimethylformamide (DMF)and, according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup is achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (Step 1). The selectivedeprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Theresin is then washed with CHCl₃ (6×5 mL) 20% HOAc in DCM (6×5 mL), DCM(6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for theaddition of the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin is washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide is cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The product is purified bypreparative reverse phase HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desired DACin >95% purity, as determined by RP-HPLC.

Example 40

Modification of Dyn A 2-17-NH₂ at the N-terminus Glycine—Synthesis ofMPA-AEA₃-Dyn A 2-17-NH₂

(MPA-AEA-AEA-AEA)-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Glu

Using automated peptide synthesis, the following protected amino acidsand maleimide were sequentially added to Rink Amide MBHA resin:Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Trp(Boc)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-AEA-OH,Fmoc-AEA-OH, Fmoc-AEA-OH, and MPA. The target dynorphin analog was thenremoved from the resin; the product was isolated by precipitation andpurified by preparative HPLC to afford the desired product as a paleyellow solid upon lyophilization in a 32% yield. Anal. HPLC indicatedproduct to be >95% pure with R_(f)=33.44 min. ESI-MS m/z forC₁₀₉H₁₇₂N₃₅O₂₉ (MH⁺), calcd 2436.8, found MH³⁺ 813.6.

Example 42

Modification of Kringle-5 at the ε-Amino Group of the Added N-TerminusLysine Residue

Preparation of (Nε-MPA)-Lys-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH₂.2TFA

Solid phase peptide synthesis of a modified Kringle-5 analog on a 100μmole scale was performed using manual solid-phase synthesis, a SymphonyPeptide Synthesizer and Fmoc protected Rink Amide MBHA. The followingprotected amino acids are sequentially added to resin: Fmoc-Tyr(tBu)-OH,Fmoc-Asp(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Lys(Aloc)-OH. They were dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N,N,N′N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group is achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). Theselective deprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Theresin is then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM(6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for theaddition of the 3maleimidopropionic acid (Step 3). Between everycoupling, the resin is washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide is cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The product is purified bypreparative reverse phase HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desired DACin >95% purity, as determined by RP-HPLC.

Example 43

Modification of Kringle-5 at the N-Terminus Proline

Preparation of (MPA-AEEA)-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-NH₂.2TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH (step 1). Thedeprotection of the terminal Fmoc group is accomplished using 20%piperidine (Step 2) followed by the coupling of Fmoc-AEEA. Deprotectionof the resulting Fmoc-AEEA-peptide with piperidine 20% in DMF allow forthe subsequent addition of the 3-MPA (Step 3). Resin cleavage andproduct isolation was performed using 86% TFA/5% TIS/5% H₂O/2%thioanisole and 2% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product was purified by preparative reversed phasedHPLC using a Varian (Rainin) preparative binary HPLC system using aDynamax C₁₈, 60 Å, 8 μm, 21 mm×25 cm column equipped with a Dynamax C₁₈,60 Å, 8 μm guard module, 21 mm×25 cm column and UV detector (VarianDynamax UVD II) at λ 214 and 254 nm. The product had >95% purity asdetermined by RP-HPLC mass spectrometry using a Hewlett PackardLCMS-1100 series spectrometer equipped with a diode array detector andusing electro-spray ionization.

Example 44

Modification of Kringle-5 at the N-Terminus Proline

Preparation of (MPA)-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-NH₂.2TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH (step 1). Thedeprotection of the terminal Fmoc group is accomplished using 20%piperidine (Step 2) followed by the coupling of 3-MPA (Step 3). Resincleavage and product isolation was performed using 86% TFA/5% TIS/5%H₂O/2% thioanisole and 2% phenol, followed by precipitation by dry-icecold Et₂O (Step 4). The product was purified by preparative reversedphased HPLC using a Varian (Rainin) preparative binary HPLC system usinga Dynamax C₁₈, 60 Å, 8 μm, 21 mm×25 cm column equipped with a DynamaxC₁₈, 60 Å, 8 μm guard module, 21 mm×25 cm column and UV detector (VarianDynamax UVD II) at λ 214 and 254 nm. The product had >95% purity asdetermined by RP-HPLC mass spectrometry using a Hewlett PackardLCMS-1100 series spectrometer equipped with a diode array detector andusing electro-spray ionization

Example 45

Modification of Kringle-5 at the N-Terminus Tyrosine

Preparation of(MPA-AEEA)-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH₂.2TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)OH (Step 1). Thedeprotection of the terminal Fmoc group is accomplished using 20%piperidine (Step 2) followed by the coupling of Fmoc-AEEA. Deprotectionof the resulting Fmoc-AEEA-peptide with piperidine 20% in DMF allow forthe subsequent addition of the 3-MPA (Step 3). Resin cleavage andproduct isolation was performed using 86% TFA/5% TIS/5% H₂O/2%thioanisole and 2% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product was purified by preparative reversed phasedHPLC using a Varian (Rainin) preparative binary HPLC system using aDynamax C₁₈, 60 Å, 8 μm, 21 mm×25 cm column equipped with a Dynamax C₁₈,60 Å, 8 μm guard module, 21 mm×25 cm column and UV detector (VarianDynamax UVD II) at λ 214 and 254 nm. The product had >95% purity asdetermined by RP-HPLC mass spectrometry using a Hewlett PackardLCMS-1100 series spectrometer equipped with a diode array detector andusing electro-spray ionization.

Example 46

Modification of Kringle-5 at the N-Terminus Tyrosine

Preparation of(MPA)-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH₂.2TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)OH (Step 1). Thedeprotection of the terminal Fmoc group is accomplished using 20%piperidine (Step 2) followed by the coupling of 3-MPA (Step 3). Resincleavage and product isolation was performed using 86% TFA/5% TIS/5%H₂O/2% thioanisole and 2% phenol, followed by precipitation by dry-icecold Et₂O (Step 4). The product was purified by preparative reversedphased HPLC using a Varian (Rainin) preparative binary HPLC system usinga Dynamax C₁₈, 60 Å. 8 μm, 21 mm×25 cm column equipped with a DynamaxC₁₈, 60 Å, 8 μm guard module, 21 mm×25 cm column and UV detector (VarianDynamax UVD II) at λ 214 and 254 nm. The product had >95% purity asdetermined by RP-HPLC mass spectrometry using a Hewlett PackardLCMS-1100 series spectrometer equipped with a diode array detector andusing electro-spray ionization.

Example 47

Modification of Kringle-5 at the N-Terminus Arginine

Preparation of(MPA-AEEA)-Arg-Asn-Pro-Asp-Gly-Asp-Gly-Pro-Trp-Ala-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH₂.3TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Ala-OH,Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH,Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH (step 1). The deprotection of theterminal Fmoc group is accomplished using 20% piperidine (Step 2)followed by the coupling of Fmoc-AEEA. Deprotection of the resultingFmoc-AEEA-peptide with piperidine 20% in DMF allow for the subsequentaddition of the 3-MPA (Step 3). Resin cleavage and product isolation wasperformed using 86% TFA/5% TIS/5% H₂O/2% thioanisole and 2% phenol,followed by precipitation by dry-ice cold Et₂O (Step 4). The product waspurified by preparative reversed phased HPLC using a Varian (Rainin)preparative binary HPLC system using a Dynamax C₁₈, 60 Å, 8 μm, 21 mm×25cm column equipped with a Dynamax C₁₈, 60 Å, 8 μm guard module, 21 mm×25cm column and UV detector (Varian Dynamax UVD II) at λ 214 and 254 nm.The product had >95% purity as determined by RP-HPLC mass spectrometryusing a Hewlett Packard LCMS-1100 series spectrometer equipped with adiode array detector and using electro-spray ionization.

Example 48

Modification of Kringle-5 at the N-Terminus Arginine

Preparation of(MPA)-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-Ala-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH₂.3TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Ala-OH,Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH,Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH (Step 1). The deprotection of theterminal Fmoc group is accomplished using 20% piperidine (Step 2)followed by the coupling of 3-MPA (Step 3). Resin cleavage and productisolation was performed using 86% TFA/5% TIS/5% H₂O/2% thioanisole and2% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct was purified by preparative reversed phased HPLC using a Varian(Rainin) preparative binary HPLC system using a Dynamax C₁₈, 60 Å, 8 μm,21 mm×25 cm column equipped with a Dynamax C₁₈, 60 Å, 8 μm guard module,21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ 214 and254 nm. The product had >95% purity as determined by RP-HPLC massspectrometry using a Hewlett Packard LCMS-1100 series spectrometerequipped with a diode array detector and using electro-spray ionization.

Example 49

Modification of Kringle-5 at the N-Terminus Arginine

Preparation of(MPA-AEEA)-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-NH₂.TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH,Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH (step 1). The deprotection of theterminal Fmoc group is accomplished using 20% piperidine (Step 2)followed by the coupling of Fmoc-AEEA. Deprotection of the resultingFmoc-AEEA-peptide with piperidine 20% in DMF allow for the subsequentaddition of the 3-MPA (Step 3). Resin cleavage and product isolation wasperformed using 86% TFA/5% TIS/5% H₂O/2% thioanisole and 2% phenol,followed by precipitation by dry-ice cold Et₂O (Step 4). The product waspurified by preparative reversed phased HPLC using a Varian (Rainin)preparative binary HPLC system using a Dynamax C₁₈, 60 Å, 8 μm, 21 mm×25cm column equipped with a Dynamax C₁₈, 60 Å, 8 μm guard module, 21 mm×25cm column and UV detector (Varian Dynamax UVD II) at λ 214 and 254 nm.The product had >95% purity as determined by RP-HPLC mass spectrometryusing a Hewlett Packard LCMS-1100 series spectrometer equipped with adiode array detector and using electro-spray ionization.

Example 50

Modification of Kringle-5 at the N-Terminus Arginine

Preparation of (MPA)-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pro-Trp-NH₂.TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH,Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH (Step 1). The deprotection of theterminal Fmoc group is accomplished using 20% piperidine (Step 2)followed by the coupling of 3-MPA (Step 3). Resin cleavage and productisolation was performed using 86% TFA/5% TIS/5% H₂O/2% thioanisole and2% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct was purified by preparative reversed phased HPLC using a Varian(Rainin) preparative binary HPLC system using a Dynamax C₁₈, 60 Å, 8 μm,21 mm×25 cm column equipped with a Dynamax C₁₈, 60 Å, 8 μm guard module,21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ 214 and254 nm. The product had >95% purity as determined by RP-HPLC massspectrometry using a Hewlett Packard LCMS-1100 series spectrometerequipped with a diode array detector and using electro-spray ionization.

Example 51

Modification of Kringle-5 at the N-Terminus Arginine

Preparation of (MPA-AEEA)-Arg-Lys-Leu-Tyr-Asp-Tyr-NH₂.2TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH (Step 1). The deprotection of theterminal Fmoc group is accomplished using 20% piperidine (Step 2)followed by the coupling of Fmoc-AEEA. Deprotection of the resultingFmoc-AEEA-peptide with piperidine 20% in DMF allow for the subsequentaddition of the 3-MPA (Step 3). Resin cleavage and product isolation wasperformed using 86% TFA/5% TIS/5% H₂O/2% thioanisole and 2% phenol,followed by precipitation by dry-ice cold Et₂O (Step 4). The product waspurified by preparative reversed phased HPLC using a Varian (Rainin)preparative binary HPLC system using a Dynamax C₁₈, 60 Å, 8 μm, 21 mm×25cm column equipped with a Dynamax C₁₈, 60 Å, 8 μm guard module, 21 mm×25cm column and UV detector (Varian Dynamax UVD II) at λ 214 and 254 nm.The product had >95% purity as determined by RP-HPLC mass spectrometryusing a Hewlett Packard LCMS-1100 series spectrometer equipped with adiode array detector and using electrospray ionization

Example 52

Modification of Kringle-5 at the N-Terminus Arginine

Preparation of (MPA)-Arg-Lys-Leu-Tyr-Asp-Tyr-NH₂.2TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH (Step 1). The deprotection of theterminal Fmoc group is accomplished using 20% piperidine (Step 2)followed by the coupling of 3-MPA (Step 3). Resin cleavage and productisolation was performed using 86% TFA/5% TIS/5% H₂O/2% thioanisole and2% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct was purified by preparative reversed phased HPLC using a Varian(Rainin) preparative binary HPLC system using a Dynamax C₁₈, 60 Å, 8 μm,21 mm×25 cm column equipped with a Dynamax C₁₈, 60 Å, 8 μm guard module,21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ 214 and254 nm. The product had >95% purity as determined by RP-HPLC massspectrometry using a Hewlett Packard LCMS-1100 series spectrometerequipped with a diode array detector and using electro spray ionization.

Example 53

Modification of Kringle-5 at the N-Terminus Proline

Preparation of (MPA-AEEA)-Pro-Arg-Lys-Leu-Tyr-Asp-NH₂.2TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH (step 1). The deprotection of the terminalFmoc group is accomplished using 20% piperidine (Step 2) followed by thecoupling of Fmoc-AEEA. Deprotection of the resulting Fmoc-AEEA-peptidewith piperidine 20% in DMF allow for the subsequent addition of the3-MPA (Step 3). Resin cleavage and product isolation was performed using86% TFA/5% TIS/5% H₂O/2% thioanisole and 2% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The product was purified bypreparative reversed phased HPLC using a Varian (Rainin) preparativebinary HPLC system using a Dynamax C₁₈, 60 Å, 8 μm, 21 mm×25 cm columnequipped with a Dynamax C₁₈, 60 Å, 8 μm guard module, 21 mm×25 cm columnand UV detector (Varian Dynamax UVD II) at λ 214 and 254 nm. The producthad >95% purity as determined by RP-HPLC mass spectrometry using aHewlett Packard LCMS-1100 series spectrometer equipped with a diodearray detector and using electro-spray ionization.

Example 54

Modification of Kringle-5 at the N-Terminus Proline

Preparation of (MPA)-Pro-Arg-Lys-Lou-Tyr-Asp-NH₂.2TFA

Using automated peptide synthesis, the following protected amino acidswere sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH (Step 1). The deprotection of the terminalFmoc group is accomplished using 20% piperidine (Step 2) followed by thecoupling of 3MPA (Step 3). Resin cleavage and product isolation wasperformed using 86% TFA/5% TIS/5% H₂O/2% thioanisole and 2% phenol,followed by precipitation by dry-ice cold Et₂O (Step 4). The product waspurified by preparative reversed phased HPLC using a Varian (Rainin)preparative binary HPLC system using a Dynamax C₁₈, 60 Å, 8 μm, 21 mm×25cm column equipped with a Dynamax C₁₈, 60 Å, 8 μm guard module, 21 mm×25cm column and UV detector (Varian Dynamax UVD II) at λ 214 and 254 nm.The product had >95% purity as determined by RP-HPLC mass spectrometryusing a Hewlett Packard LCMS-1100 series spectrometer equipped with adiode array detector and using electro-spray ionization.

3. Modification at an Internal Amino Acid

Example 55

Synthesis of Lys²⁶(ε-MPA)GLP-1(7-36)-NH₂

Solid phase peptide synthesis of a modified GLP-1 analog on a 100 μmolescale was performed manually and on a Symphony Peptide Synthesizer usingFmoc protected Rink amide MBHA resin. The following protected aminoacids are sequentially added to the resin: Fmoc-Arg(Pbf)-OH,Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH,Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH,Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH,Boc-His(Trt)-OH. They are dissolved in N,N-dimethylformamide (DMF) and,according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup is achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (Step 1). Selectivedeprotection of the Lys(Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Theresin is then washed with CHCl₃ (6×5mL), 20% HOAc in DCM (6×5mL), DCM(6×5mL), and DMF (6×5mL). The synthesis is then re-automated for theaddition of the 3-maleimidopropionic acid (Step 3). Resin cleavage andproduct isolation is performed using 86% TFA/5% TIS/5% H₂O/2%thioanisole and 2% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product is purified by preparative reversed phaseHPLC using a Varian (Rainin) preparative binary HPLC system using aDynamax C₁₈, 60 Å, 8 μm, 21 mm×25 cm column equipped with a Dynamax C₁₈,60 Å, 8 μm guard module, 21 mm×25 cm column and UV detector (VarianDynamax UVD II) at λ 214 and 254 nm to afford the desired DAC in >95%purity, as determined by RP-HPLC. These steps are illustrated in theschematic diagram below.

D. Preparation of Modified Peptides from Peptides Containing One FreeCysteine

Preparation of maleimido peptides from therapeutic peptides containingone free Cysteine is exemplified by the synthesis of peptides asdescribed below. The peptide may be modified at the N-terminus, theC-terminus, or at an amino acid located between the N-terminus and theC-terminus.

Preparation of maleimido peptides from peptides containing multipleprotected functional groups and multiple Cysteine residues all with onefree Cysteine residues (i.e. all Cysteine residues, except one, are tiedup as disulfides). Linking from an internal amino acid in the naturalsequence as in Example 5. The free Cysteine residue must be capped orreplaced with another amino acid (e.g. Alanine, Methionine, etc.).

Where the peptide contains one cysteine, the cysteine must stay cappedafter addition of the maleimide. If the cysteine is involved in bindingsite, assessment has to be made of how much potency is lost is cysteineis capped by a protecting group. If the cysteine can stay capped, thenthe synthetic path is similar to example (i) above. Examples oftherapeutic peptides that contain one cystein include G_(α) (the alphasubunit of Gtherapeutic peptide binding protein), the 724-739 fragmentof rat brain nitric oxide synthase blocking peptide, the alpha subunit1-32 fragment of human [Tyr0] inhibin, the 254-274 fragment of HIVenvelope protein, and P34cdc2 kinase fragment.

1. Modification at the N-Terminus

Example 56

Modification of Inhibin Peptide at the Added N-Terminus Lysine

Preparation of(Nε-MPA)-Lys-Tyr-Ser-Thr-Pro-Leu-Met-Ser-Trp-Pro-Trp-Ser-Pro-Ser-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala-Ala-His-Ala-Asn-Cys-His-Arg

Solid phase peptide synthesis of a modified inhibin peptide analog on a100 μmole scale is performed using manual solid-phase synthesis, aSymphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. Thefollowing protected amino acids are sequentially added to resin:Fmoc-Arg(Pbf)-OH, Fmoc-His(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Ala-OH, Fmoc-His(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Pro-OH,Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH,Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Pro-OH,Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Met-OH, Fmoc-Leu-OH,Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Lys(Aloc)-OH. They are dissolved in N,N-dimethylformamide (DMF)and, according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup is achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (Step 1). The selectivedeprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Theresin is then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM(6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for theaddition of the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin is washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide is cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The product is purified bypreparative reverse phase HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desired DACin >95% purity, as determined by RP-HPLC.

Example 57

Modification of RSV Antifusogenic Peptide at the N-Terminus

Preparation of3-(MPA)-Val-Ile-Thr-Ile-Glu-Leu-Ser-Asn-Ile-Lys-Glu-Asn-Lys-Cys-Asn-Glu-Ala-Lys-Val-Lys-Leu-Ile-Lys-Glu-Glu-Leu-Asp-Lys-Tyr-Lys-Asn-Ala-Val

Initially, (Cysteine (Cys) was replaced with Methionine (Met) within thenative sequence. Solid phase peptide synthesis of a modified anti RSVanalog on a 100 μmole scale was performed on a Symphony PeptideSynthesizer using Fmoc protected Rink Amide MBHA resin, Fmoc protectedamino acids, O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) in N,N-dimethylformamide (DMF) solution andactivation with N-methyl morpholine (NMM), and piperidine deprotectionof Fmoc groups (Step 1). The deprotection of the terminal Fmoc group isaccomplished using 20% piperidine (Step 2) followed by the coupling of3-MPA (Step 3). Resin cleavage and product isolation was performed using85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitationby dry-ice cold Et₂O (Step 4). The product is purified by preparativereverse phase HPLC using a Varian (Rainin) preparative binary HPLCsystem: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045%TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μphenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II)at λ 214 and 254 nm to afford the desired DAC in >95% purity, asdetermined by RP-HPLC. These steps are illustrated by the schematicdiagram below.

2. Modification at the C-Terminus

Example 58

Modification of Inhibin Peptide at the Added C-Terminus Lysine

Preparation of(Nε-MPA)-Lys-Cys-Asn-Leu-Lys-Glu-Asp-Gly-Ile-Ser-Ala-Ala-Lys-Asp-Val-Lys

Solid phase peptide synthesis of a modified inhibin peptide analog on a100 μmole scale is performed using manual solid-phase synthesis, aSymphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. Thefollowing protected amino acids are sequentially added to resin:Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Asp(tBu)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Gly-OH,Fmoc-Asp(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH,Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Aloc)-OH, They aredissolved in N,N-dimethylformamide (DMF) and, according to the sequence,activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group is achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (Step 1). Theselective deprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Theresin is then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM(6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for theaddition of the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin is washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide is cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The product is purified bypreparative reverse phase HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desired DACin >95% purity, as determined by RP-HPLC.

Example 59

Modification of RSV Fusogenic Peptide at the C-Terminus

Val-Ile-Thr-Ile-Glu-Leu-Ser-Asn-Ile-Lys-Glu-Asn-Lys-Cys-Asn-Gly-Ala-Lys-Val-Lys-Leu-Ile-Lys-Gln-Glu-Leu-Asp-Lys-Tyr-Asn-Ala-Val-(AEEA.MPA)

Preparation of maleimido peptides from peptides containing multipleprotected functional groups and one cysteine is exemplified by thesynthesis of a modified RSV fusogenic peptide. The modified RSVfusogenic peptide was synthesized by linking off the C-terminus by theaddition of a lysine residue to the natural peptide sequence asillustrated by the schematic diagram below. In cases where a cysteineresidue is contained within the peptide sequence and is not essentiallyto the biological activity of the peptide, this residue must be replacedwith another amino acid (e.g. alanine, methionine, etc.). In thefollowing synthesis, the cysteine (Cys) was replaced with a methionine(Met) within the native RSV sequence.

Solid phase peptide synthesis of the maleimido RSV fusogenic peptide ona 100 μmole scale is performed using manual solid-phase synthesis and aSymphony Peptide Synthesizer using Fmoc protected Rink Amide MBHA resin,Fmoc protected amino acids,O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) in N,N-dimethylformamide (DMF) solution and activation withN-methyl morpholine (NMM), and piperidine deprotection of Fmoc groups(Step 1). The selective deprotection of the Lys(Aloc) group is performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h(Step 2). The resin is then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM(6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is thenre-automated for the addition of 3-maleimidopropionic acid (Step 3).Resin cleavage and product isolation is performed using 85% TFA/5%TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-icecold Et₂O (Step 4). The product is purified by preparative reverse phaseHPLC using a Varian (Rainin) preparative binary HPLC system: gradientelution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B))over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ 214 and 254nm to afford the desired DAC in >95% purity, as determined by RP-HPLC.These steps are illustrated in the schematic diagram below.

3. Modification at an Internal Amino Acid

Example 60

Modification of Gα Peptide atCys-Asn-Leu-Lys-Glu-Asp-Gly-Ile-Ser-Ala-Ala-Lys-Asp-Val

Preparation of maleimido peptides from peptides containing multipleprotected functional groups and one cysteine is exemplified by thesynthesis of a modified Gα peptide. The modified Gα peptide issynthesized by linking at an internal amino acid as described below.

In cases where a cysteine residue is contained within the peptidesequence and is not essentially to the biological activity of thepeptide, this residue must be capped or replaced with another amino acid(e.g. alanine, methionine, etc.). Solid phase peptide synthesis of themodified Gα peptide on a 100 μmole scale is performed using manualsolid-phase synthesis and a Symphony Peptide Synthesizer using Fmocprotected Rink Amide MBHA resin, Fmoc protected amino acids,O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate(HBTU) in N,N-dimethylformamide (DMF) solution and activation withN-methyl morpholine (NMM), and piperidine deprotection of Fmoc groups(Step 1). The selective deprotection of the Lys(Aloc) group is performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h(Step 2). The resin is then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM(6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is thenre-automated for the addition of 3-maleimidopropionic acid (Step 3).Resin cleavage and product isolation is performed using 85% TFA/5%TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-icecold Et₂O (Step 4). The product is purified by preparative reversedphased HPLC using a Varian (Rainin) preparative binary HPLC system:gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA inCH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μphenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II)at λ 214 and 254 nm to afford the desired DAC in >95% purity, asdetermined by RP-HPLC.

E. Preparation of Modified Peptides from Peptides Containing TwoCysteines In Disulfide Bridge

Where the peptide contains two cysteines as a disulfide bridge, thepeptide is cleaved from the support resin before addition of themaleimide. We need to add a Lys protected with a Mtt group in order toselectively deprotect the lysine in presence of other t-Boc protectedlysine. All protecting groups are present except at the carboxy terminus(which stays unprotected due to cleavage from the support resin) and atthe two cysteines, which need to be deprotected when peptide is cleavedfrom resin. Mild air oxidation yield the disulfide bridge, and thepeptide can be purified at that stage. Solution phase chemistry is thenrequired to activate the C-terminus in presence of the disulfide bridgeand add the maleimide (through an amino-alkyl-maleimide) to theC-terminus. The peptide is then fully deprotected. Examples oftherapeutic peptides that contain two cysteins as a disulfide bridgeinclude human osteocalcin 1-49, human diabetes associated peptide, the5-28 fragment of human/canine atrial natriuretic peptide, bovinebactenecin, and human [Tyr0]-cortistatin 29.

Preparation of maleimido peptides from therapeutic peptides containingtwo Cysteines in a disulfide bridge is exemplified by the synthesis ofpeptides as described below. The peptide may be modified at theN-terminus, the C-terminus, or at an amino acid located between theN-terminus and the C-terminus.

1. Modification at the N-Terminus

Example 61

Modification of TH-1 Peptide at N-Terminus

Preparation of(Nε-MPA)NH₂-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Cys

Preparation of thiol cyclized maleimido peptides from peptidescontaining multiple protected functional groups and no free cysteineresidues (i.e. all cysteine residues are tied up as disulfide bridges)is illustrated by the synthesis of a modified TH-1 peptide.

Solid phase peptide synthesis of the modified TH-1 peptide on a 100μmole scale was performed manually and on a Symphony Peptide Synthesizerusing Fmoc protected Rink Amide MBHA resin, Fmoc protected amino acids,O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) in N,N-dimethylformamide (DMF) solution and activation withN-methyl morpholine (NMM), and piperidine deprotection of Fmoc groups(Step 1). The removal of the Acm group and resulting oxidation of thetwo Cys residues to form the cyclized on resin DAC was accomplishedusing TI(CF₃CO)₂ (Step 2). The deprotection of the terminal Fmoc groupis accomplished using 20% piperidine followed by the coupling of 3-MPA(Step 3). Resin cleavage and product isolation was performed using 86%TFA/5% TIS/5% H₂O/2% thioanisole and 2% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The product was purified bypreparative reverse phase HPLC using a Varian (Rainin) preparativebinary HPLC system using a Dynamax C₁₈, 60 Å, 8 μm, 21 mm×25 cm columnequipped with a Dynamax C₁₈, 60 Å, 8 μm guard module, 21 mm×25 cm columnand UV detector (Varian Dynamax UVD II) at λ 214 and 254 nm. The producthad >95% purity as determined by RP-HPLC mass spectrometry using aHewlett Packard LCMS-1100 series spectrometer equipped with a diodearray detector and using electro-spray ionization, ESI-MS m/z forC₆₆H₉₅N₂₀O₂₆S₂ (MH⁺), 1646.8. Found: 1646.7. These steps are illustratedin the schematic diagram below.

Example 62

Synthesis of N-MPA-Ser¹-Somatostatin-28

Solid phase peptide synthesis of the DAC:Somatostatin-28 analog on a 100μmole scale is performed manually and on a Symphony Peptide Synthesizerusing Fmoc protected Rink amide MBHA resin. The following protectedamino acids are sequentially added to the resin: Fmoc-Cys(Acm)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH,Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH,Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Met-OH, Fmoc-Ala-OH,Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH. They are dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group is achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (Step 1).Removal of the Acm groups and resulting oxidation of the two Cysresidues to form the disulfide bridge is accomplished using iodine (Step2). Deprotection of the terminal Fmoc group is accomplished using 20%piperidine followed by the coupling of 3-MPA (Step 3). Resin cleavageand product isolation is performed using 86% TFA/5% TIS/5% H₂O/2%thioanisole and 2% phenol, followed by precipitation by dry-ice coldEt₂O (Step 4). The product is purified by preparative reversed phaseHPLC using a Varian (Rainin) preparative binary HPLC system using aDynamax C₁₈, 60 Å, 8 μm, 21 mm×25 cm column equipped with a Dynamax C₁₈,60 Å, 8 μm guard module, 21 mm×25 cm column and UV detector (VarianDynamax UVD II) at λ 214 and 254 nm to afford the desired DAC in >95%purity, as determined by RP-HPLC. These steps are illustrated in theschematic diagram below.

2. Modification at the C-Terminus

Example 63

Synthesis of Somatostatin-28-EDA-MPA

Solid phase peptide synthesis of the DAC:Somatostatin-28 analog on a 100μmole scale is performed manually and on a Symphony Peptide Synthesizerusing SASRIN (super acid sensitive resin). The following protected aminoacids are sequentially added to the resin: Fmoc-Cys(Acm)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH,Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH,Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Met-OH, Fmoc-Ala-OH,Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Ala-OH, Boc-Ser(tBu)-OH. They are dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group is achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (Step 1). Thefully protected peptide is cleaved from the resin by treatment with 1%TFA/DCM (Step 2). Removal of the Acm groups and resulting oxidation ofthe two Cys residues to form the disulfide bridge is accomplished usingiodine (Step 3). Ethylenediamine and 3-maleimidopropionic acid are thensequentially added to the free C-terminus (Step 4). The protectinggroups are then cleaved and the product isolated using 86% TFA/5% TIS/5%H₂O/2% thioanisole and 2% phenol, followed by precipitation by dry-icecold Et₂O (Step 5). The product is purified by preparative reversedphase HPLC using a Varian (Rainin) preparative binary HPLC system usinga Dynamax C₁₈, 60 Å, 8 μm, 21 mm×25 cm column equipped with a DynamaxC₁₈, 60 Å, 8 μm guard module, 21 mm×25 cm column and UV detector (VarianDynamax UVD II) at λ 214 and 254 nm to afford the desired DAC in >95%purity, as determined by RP-HPLC. These steps are illustrated in theschematic diagram below.

3. Modification at an Internal Amino Acid

Example 64

Synthesis of Lys¹⁴(ε-MPA)-Somatostatin-28

Solid phase peptide synthesis of the DAC:Somatostatin-28 analog on a 100μmole scale is performed manually and on a Symphony Peptide Synthesizerusing Fmoc protected Rink amide MBHA resin. The following protectedamino acids are sequentially added to the resin: Fmoc-Cys(Acm)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH,Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH,Fmoc-Ala-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Met-OH, Fmoc-Ala-OH,Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH. They are dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group is achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (Step 1).Removal of the Acm groups and resulting oxidation of the two Cysresidues to form the disulfide bridge is accomplished using iodine (Step2). Selective deprotection of the Lys(Aloc) group is performed manuallyand accomplished by treating the resin with a solution of 3 eq ofPd(PPh₃)₄ dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step3). The resin is then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automatedfor the addition of the 3-maleimidopropionic acid (Step 4). Resincleavage and product isolation is performed using 86% TFA/5% TIS/5%H₂O/2% thioanisole and 2% phenol, followed by precipitation by dry-icecold Et₂O (Step 5). The product is purified by preparative reversedphase HPLC using a Varian (Rainin) preparative binary HPLC system usinga Dynamax C₁₈, 60 Å, 8 μm, 21 mm×25 cm column equipped with a DynamaxC₁₈, 60 Å, 8 μm guard module, 21 mm×25 cm column and UV detector (VarianDynamax UVD II) at λ 214 and 254 nm to afford the desired DAC in >95%purity, as determined by RP-HPLC. These steps are illustrated in thefollowing schematic diagram.

F. Preparation of Modified Peptides from Peptides Containing MultipleCysteines1. Modification at the N-Terminus

Example 65

Synthesis of N-MPA-Cys¹-Endothelin-1 (1-21)-OH

Solid phase peptide synthesis of a modified Endothelin-1 analog on a 100μmole scale is performed manually and on a Symphony Peptide Synthesizerusing Fmoc protected Rink Amide MBHA resin. The following protectedamino acids are sequentially added to the resin: Fmoc-Trp(Boc)-OH,Fmoc-Ile-OH, Fmoc-Ile-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH,Fmoc-His(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Val-OH, Fmoc-Cys(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Cys(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cys(Acm)-OH.They are dissolved in N,N-dimethylformamide (DMF) and, according to thesequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroups is achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (Step 1). The removal of theAcm groups and resulting oxidation of the first two Cys residues to formthe first disulfide bridge on resin is accomplished using iodine (Step2). The removal of the tBu groups and resulting oxidation of the othertwo Cys residues to form the second disulfide bridge on resin isaccomplished using thallium (III) trifluoroacetate (Step 3). Thedeprotection of the terminal Fmoc group is accomplished using 20%piperidine followed by the coupling of 3-MPA (Step 4). Resin cleavageand product isolation is performed using 86% TFA/5% TIS/5% H₂O/2%thioanisole and 2% phenol, followed by precipitation by dry-ice coldEt₂O (Step 5). The product is purified by

preparative reverse phase HPLC using a Varian (Rainin) preparativebinary HPLC system using a Dynamax C₁₈, 60 Å, 8 μm, 21 mm×25 cm columnequipped with a Dynamax C₁₈, 60 Å, 8 μm guard module, 21 mm×25 cm columnand UV detector (Varian Dynamax UVD II) at λ 214 and 254 nm to affordthe desired DAC in >95% purity, as determined by RP-HPLC. These stepsare illustrated in the schematic diagram above.2. Modification at the C-Terminus

Example 66

Synthesis of Endothelin-1 (1-21)Lys²²-(Nε-MPA)-OH

Solid phase peptide synthesis of a modified Endothelin-1 analog on a 100μmole scale is performed manually and on a Symphony Peptide Synthesizerusing Fmoc protected Rink Amide MBHA resin. The following protectedamino acids are sequentially added to the resin: Fmoc-Lys(Aloc)-OH,Fmoc-Trp(Boc)-OH, Fmoc-Ile-OH, Fmoc-Ile-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Phe-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Cys(tBu)-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Leu-OH,Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cys(tBu)-OH, Fmoc-Ser(tBu)-OH,Boc-Cys(Acm)-OH. They are dissolved in N,N-dimethylformamide (DMF) and,according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup is achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (Step 1). The removal of theAcm groups and resulting oxidation of the first two Cys residues to formthe first disulfide bridge on resin is accomplished using iodine (Step2). The removal of the tBu groups and resulting oxidation of the othertwo Cys residues to form the second disulfide bridge on resin isaccomplished using thallium (III) trifluoroacetate (Step 3). Selectivedeprotection of the Lys(Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh3)4dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 4). Theresin is then washed with CHCl3 (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM(6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for theaddition of the 3-maleimidopropionic acid (Step 5). Resin cleavage andproduct isolation is performed using 86% TFA/5% TIS/5% H₂O/2%thioanisole and 2% phenol, followed by precipitation by dry-ice coldEt₂O (Step 5). The product is purified by preparative reverse phase HPLCusing a Varian (Rainin) preparative binary HPLC system using a DynamaxC₁₈, 60 Å, 8 μm, 21 mm×25 cm column equipped with a Dynamax C₁₈, 60 Å, 8μm guard module, 21 mm×25 cm column and UV detector (Varian Dynamax UVDII) at λ 214 and 254 nm to afford the desired DAC in >95% purity, asdetermined by RP-HPLC. These steps are illustrated in the schematicdiagram below.

3. Modification at an Internal Amino Acid

Example 67

Synthesis of Lys⁴(Nε-MPA)Sarafotoxin B(1-21)-OH

Solid phase peptide synthesis of a modified Sarafotoxin-B analog on a100 μmole scale is performed manually and on a Symphony PeptideSynthesizer using Fmoc protected Rink Amide MBHA resin. The followingprotected amino acids are sequentially added to the resin:Fmoc-Trp(Boc)-OH, Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH,Fmoc-Gln(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Phe-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Cys(tBu)-OH, Fmoc-Glu(OtBu)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Met-OH,Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Cys(tBu)-OH,Fmoc-Ser(tBu)-OH, Boc-Cys(Acm)-OH. They are dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group is achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (Step 1). Theremoval of the Acm groups and resulting oxidation of the first two Cysresidues to form the first disulfide bridge on resin is accomplishedusing iodine (Step 2). The removal of the tBu groups and resultingoxidation of the other two Cys residues to form the second disulfidebridge on resin is accomplished using thallium (III) trifluoroacetate(Step 3). Selective deprotection of the Lys(Aloc) group is performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh3)4 dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h(Step 4). The resin is then washed with CHCl3 (6×5 mL), 20% HOAc in DCM(6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is thenre-automated for the addition of the 3-maleimidopropionic acid (Step 5).Resin cleavage and product isolation is performed using 86% TFA/5%TIS/5% H₂O/2% thioanisole and 2% phenol, followed by precipitation bydry-ice cold Et₂O (Step 5). The product is purified by preparativereverse phase HPLC using a Varian (Rainin) preparative binary HPLCsystem using a Dynamax C₁₈, 60 Å, 8 μm, 21 mm×25 cm column equipped witha Dynamax C₁₈, 60 Å, 8 μm guard module, 21 mm×25 cm column and UVdetector (Varian Dynamax UVD II) at λ 214 and 254 nm to afford thedesired DAC in >95% purity, as determined by RP-HPLC. These steps areillustrated in the schematic diagram below.

F. Peptide Stability Assays

Example 68

Peptide Stability Assay of K5

A peptide stability assay was performed.(MPA)-Pro-Arg-Lys-Leu-Tyr-Asp-Lys-NH₂. 2TFA was synthesized as describedabove and was identified MPA-K5. The non-modified counterpart peptidePro-Arg-Lys-Leu-Tyr-Asp-Lys was also synthesized as described in Example20 without the addition of 3-MPA and identified as K5.

K5 (MW1260.18, 918.12 freebase) was prepared as a 100 mM stock solutionin water. MPA-K5(MW=1411.17, 1069.11 freebase) was prepared as a 100 mMstock solution in water. Human Serum Albumin (HSA) was obtained as a 25%solution (ca 250 mg/ml, 3.75 mM) as Albutein® available from AlphaTherapeutic. Human plasma was obtained from Golden West Biologicals.

(1) Stability of K5 in Human Plasma

K5 was prepared as a 1 μM solution and dissolved in 25% human serumalbumin. The mixture was then incubated at 37° C. in the presence ofhuman plasma to final concentration of 160 mM K5. Aliquots of 100 μlwere withdrawn from the plasma at 0, 4 hours and 24 hours. The 100 μlaliquots were mixed with 100 μl of blocking solution (5 vol. 5%ZnSO₄/3vol. Acetonitrile/2 vol. Methanol) in order to precipitate all proteins.The sample was centrifuged for 5 min at 10,000 g and the supernatantcontaining the peptide was recovered and filtered through a 0.22 μmfilter. The presence of free intact K5 peptide was assayed by theHPLC/MS. The results are presented below. The HPLC parameters fordetection of K5 peptide in serum were as follows.

The HPLC method was as follows: A Vydac C18 250×4.6 mm, 5μ particle sizecolumn was utilized. The column temperature was 30° C. with a flow rateof 0.5 ml/min. Mobile Phase A was 0.1% TFA/water. Mobile Phase B was0.1% TFA/acetonitrite. The injection volume was 10 μl.

The gradient was as follows:

Time (Minutes) % A % B 0 95 5 20 70 30 25 10 90 30 10 90 35 95 5 45 95 5

The proteins were detected at 214, 254 and 334 nm. For mass spectralanalysis, the ionization mode was API-electrospray (positive mode) at anM/Z range of 300 to 2000. The gain was 3.0, fragmentor 120 v, threshold20, stepsize 0.1. The gas temp was 350° C. and the drying gas volume was10.0 l/min. The Neb pressure was 24 psi and the Vcap was 3500V. The HPLCmethod was as follows: A Vydac C18 250×4.6 mm, 5μ particle size columnwas utilized. The column temperature was 30° C. with a flow rate of 0.5ml/min. Mobile Phase A was 0.1% TFA/water. Mobile Phase B was 0.1%TFA/acetonitrite. The injection volume was 10 μl.

The gradient was as follows:

Time (Minutes) % A % B 0 95 5 20 70 30 25 10 90 30 10 90 35 95 5 45 95 5

The proteins were detected at 214, 254 and 334 nm. For mass spectralanalysis, the ionization mode was API-electrospray (positive mode) at anMEZ range of 300 to 2000. The gain was 3.0, fragmentor 120 v, threshold20, stepsize 0.1. The gas temp was 350° C. and the drying gas volume was10.0 l/min. The Neb pressure was 24 psi and the Vcap was 3500V.

Time % K5 peptide in Plasma  0 hrs. 100%  4 hrs.  9% 24 hrs.  0%

The results demonstrate that unmodified K5 peptide is unstable in plasmalikely as a result of protease activity.

(2) Stability of MPA-K5-HSA Conjugate in Plasma

MPA-K5 (modified K5 peptide) was incubated with 25% HSA for 2 hours atroom temperature. The MPA-K5-HSA conjugate was then incubated at 37° inthe presence of human plasma at a final concentration of 160 μm. Afterthe specific incubation period (0, 4 and 24 hours) an aliquot of 100 μlwas withdrawn and filtered through a 0.22 μm filter. The presence ofintact conjugate was assayed by HPLC-MS.

The column was an Aquapore RP-300, 250×4.6 mm, 7μ particle size. Thecolumn temperature was 50° C. The mobile phase A was 0.1% TFA/water. Themobile phase B was 0.1% TFA/acetonitrile. The injection volume was 1 μl.The gradient was as follows:

Time (minutes) % A % B Flow (ml/min) 0 56 34 0.700 1 66 34 0.700 25 58.841.2 0.700 30 50 50 0.70 35 5 95 1.00 41 5 95 1.00 45 66 34 1.00 46 6634 0.70

The peptide was detected at 214 mm for quantification. For mass spectralanalysis of the peptide, the ionization mode was API-electrospray at1280 to 1500 m/z range, gain 1.0, fragmentor 125V, threshold 100,stepsize 0.40. The gas temperature was 350° C. the drying gas was 13.0l/min. The pressure was 60 psi and the Vcap was 6000V. The results arepresented below.

Approximately 33% of circulating albumin in the bloodstream ismercaptalbumin (SH-albumin) which is not blocked by endogenoussulfhydryl compounds such as cysteine or glutathione and is thereforeavailable for reaction with maleimido groups. The remaining 66% of thecirculating albumin is capped or blocked by sulfhydryl compounds. TheHPLC MS assay permits the identification of capped-HSA, SH-albumin andK5MPA-albumin. The MPA covalently bonds to the free thiol on thealbumin. The stability of the three forms of albumin in plasma ispresented below.

Time % capped HSA % SH-Albumin % K5-MPA-HSA  0 hrs. 61.3 16.6 22.1  4hrs. 64.6 16.05 19.35 24 hrs. 63 16.8 20.2

The percentage of K5-MPA-HSA remained relatively constant throughout the24 hour plasma assay in contrast to unmodified K5 which decreased to 9%of the original amount of K5 in only 4 hours in plasma. The resultsdemonstrate that in contrast to K5 which is quite unstable in plasma,K5-MPA-HSA is quite stable from peptidase activity in plasma.

Example 70

Peptide Stability Assay of Dynorphin

In order to determine the stability of peptide conjugates in thepresence of serum peptidases the serum stability of Dyn A-(1-13)-OH, DynA-(1-13)-NH₂ and Dyn A 1-13(MPA)-NH₂ were compared. Dyn A-(1-13)-OH, DynA-(1-13)-NH₂ and Dyn A 1-13(MPA)NH₂ were synthesized as described above.The Dynorphin peptides were mixed with human heparinized plasma to afinal concentration of 4 mg/mL. After the required incubation time at37° C., 0, 20, 20, 60, 120, 180, 360 and 480 minutes) a 100 μL-aliquotwas added to 100 μL of blocking solution (5 vol. of a 5% aqueous ZnSO₄solution, 3 vol. of acetonitrile, 2 vol. of methanol) that precipitatesall proteins. After centrifugation (10,000 g for 2.5 min), clearsupernatant was recovered, filtered through a 0.45 μm filter and storedon ice until LC/MS analysis.

The samples were analyzed using an LC at 214 nm to detect the presenceof the different compounds and MS to determine the identity of thedetected compound. The integrated area % for each peak from the LCchromatogram was then plotted against time and the relative stabilitiesdetermined in human plasma.

The stability data for Dyn A-(1-1 3)-OH and Dyn A-(1-1 3)-NH₂ wereconsistent with that reported in literature: the proteolytic breakdownof the dynorphin peptides is quite rapid. Dyn A-(1-13)-OH had a halflife of about 10 minutes. Dyn A-(1-13)-NH₂ had a half life of about 30minutes. In contrast Dyn A 1-13(MPA)-NH₂ exhibited strikingstabilization in the presence of serum peptidase activity. Unmodifieddynorphin peptides are degraded within 60 minutes. In contrast, modifieddynorphin peptides (Dyn A 1-13(MPA)-NH₂) are stable from serum peptidaseactivity for up to 480 minutes.

The stability determination of the dynorphin conjugate is determined byELISA. In order to determine if the observed signal is due to adynorphin conjugate and what the conjugate is, LC mass spectrometrytralanalysis of the reaction mixture after 8 h was performed. The use ofmass spectrometry permits a determination of the molecular weight of theconjugate and allows the determination whether there are any truncatedforms of the dynorphin conjugate. Mass spectrometry of human plasmashows the two forms of albumin, the free thiol at 66436 Da and theoxidized form at 66557 Da. Also, mass spectrometry can distinguishbetween a Dyn 2-13 truncated conjugate (68046 Da) and the intact Dyn1-13 conjugate, (68207 Da) in an equal mixture.

Mass spectrometry analysis of dynorphin samples taken from the serumafter 480 minutes of exposure to the serum peptidases identifies onlythe presence of the intact conjugate (68192 Da) and not the breakdownproducts thereby demonstrating the stability of the dynorphin conjugatefrom serum peptidase activity.

TABLE 1 NATURAL AMINO ACIDS AND THEIR ABBREVIATIONS 3-Letter 1-LetterProtected Amino Name Abbreviation Abbreviation Acids Alanine Ala AFmoc-Ala-OH Arginine Arg R Fmoc-Arg(Pbf)-OH Asparagine Asn NFmoc-Asn(Trt)-OH Aspartic acid Asp D Asp(tBu)-OH Cysteine Cys CFmoc-Cys(Trt) Glutamic acid Glu E Fmoc-Glu(tBu)-OH Glutamine Gln QFmoc-Gln(Trt)-OH Glycine Gly G Fmoc-Gly-OH Histidine His HFmoc-His(Trt)-OH Isoleucine Ile I Fmoc-Ile-OH Leucine Leu L Fmoc-Leu-OHLysine Lys K Fmoc-Lys(Mtt)-OH Methionine Met M Fmoc-Met-OH PhenylalaninePhe F Fmoc-Phe-OH Proline Pro P Fmoc-Pro-OH Serine Ser SFmoc-Ser(tBu)-OH Threonine Thr T Fmoc-Thr(tBu)-OH Tryptophan Trp WFmoc-Trp(Boc)-OH Tyrosine Tyr Y Boc-Tyr(tBu)-OH Valine Val V Fmoc-Val-OH

1. A method of synthesizing a modified therapeutic peptide capable offorming a peptidase-stabilized therapeutic peptide conjugate, thepeptide comprising between 3 and 50 amino acids and having a carboxyterminal amino acid, an amino terminal amino acid, the method ofcomprising the steps of: a) synthesizing the peptide from the carboxyterminal amino acid or the amino terminal amino acid, b) sequentiallyand selectively oxidizing any pairs of cysteine residues in saidtherapeutic peptide to form disulfide bridges in said therapeuticpeptide; c) attaching a protecting group to any remaining cysteineresidues that do not form said disulfide bridges in said therapeuticpeptide; and d) coupling a reactive group to the carboxy terminal aminoacid, to the amino terminal amino acid, or to an amino acid between thecarboxy terminal amino acid and the amino terminal amino acid, whereinthe reactive group is capable of reacting with an amino group, anhydroxyl group or a thiol group on blood component to form a covalentbond therewith.
 2. A method as claimed in claim 1 wherein the reactivegroup is selected from the group consisting of succinimidyl- andmaleimido-containing groups.
 3. A method as claimed in claim 2 whereinthe reactive entity is a maleimido-containing group.
 4. A method asclaimed in claim 1, further comprising bonding a lysine residue to saidpeptide, wherein the reactive group is coupled to the peptide via saidlysine residue.
 5. A method as claimed in claim 1 wherein the reactivegroup is coupled to the carboxy terminal amino acid of the peptide.
 6. Amethod as claimed in claim 1 wherein the peptide does not contain acysteine.
 7. A method as claimed in claim 1 wherein the therapeuticpeptide contains two cysteines, the two cysteines are oxidized to form adisulfide bridge, and the reactive group is coupled to the peptide.
 8. Amethod as claimed in claim 6 wherein the peptide is synthesized from thecarboxy terminal amino acid.
 9. A method of synthesizing a modifiedtherapeutic peptide and forming a peptidase-stabilized therapeuticpeptide conjugate, the peptide comprising between 3 and 50 amino acidsand having a carboxy terminal amino acid and an amino terminal aminoacid, the method comprising the steps of synthesizing the peptide fromthe carboxy terminal amino acid, coupling a maleimido-containing group,to the carboxy terminal amino acid, the amino terminal amino acid, anamino acid between the carboxy terminal amino acid and the aminoterminal amino acid, and reacting the maleimido-containing group with athiol group on a blood component to form a covalent bond therewith. 10.A method as claimed in claim 9 wherein the maleimido-containing group iscoupled to the carboxy terminal amino acid.
 11. A method as claimed inclaim 9, further comprising bonding a lysine residue to said peptide,wherein the maleimido-containing group is coupled to the peptide viasaid lysine residue.
 12. A method as claimed in claim 9 wherein saidreacting step occurs in vivo.
 13. A method as claimed in claim 9 whereinsaid reacting step occurs ex vivo.